Anti-KC4 humanized monoclonal antibody and cells that express the antibody

ABSTRACT

An anti-KC-4 humanized monoclonal antibody that comprises the variable regions of the light and heavy chains of the anti-KC-4 murine antibody, wherein the light chain has 7 amino acids and the heavy chain has 12 amino acids of the framework regions substituted with amino acid present in equivalent positions in antibodies of a species other than munne, and the constant regions of a human antibody. The antibody may be labeled and/or glycosylated, and is presented as a composition with a carrier. The anti-KC-4 monoclonal antibody is used in diagnostic kits for cancer and in in vivo methods of imaging and treating a primary or metastasized cancer, and in vitro diagnosis and ex vivo purging neoplastic cells from a biological fluid. RNAs and DNAs encode the monoclonal antibody, and a hybrid vector carrying the nucleotides and transfected cells express the peptides.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the in vito and in vivo diagnosis and therapyof carcinomas by means of a specifically targeted humanized mousemonoclonal antibody selectively binding the human KC-4 antigen. Thehumanized anti-KC-4 mouse antibody comprises the complementaritydetermining regions (CDRs) of the variable regions of the mouse antibodyof the same specificity, and its framework regions having specific aminoacids replaced in a predetermined manner, and the constant regions of ahuman antibody. The humanized anti-KC-4 mouse antibody of this inventionis expected to elicit a lesser immunological response in humans than thewhole mouse antibody and is therefore considered suitable for in vivoadministration to humans. Polynucleotide segments encoding the humanizedantibody, a hybrid vector and a transfected host cell carrying the DNAsegments encoding the antibody are useful for preparing the peptidesdisclosed herein.

2. Description of the Background

Carcinomas result from the carcinogenic transformation of cells ofdifferent epithelia. Two of the most damaging characteristics ofcarcinomas are their uncontrolled growth and their ability to createmetastases in distant sites of the host, particularly a human host. Itis usually these distant metastases that cause serious consequences tothe host, since frequently the primary carcinoma may be, in most cases,removed by surgery. The treatment of metastatic carcinomas, that areseldom removable, depends on irradiation therapy and systemic therapiesof different natures. The systemic therapies currently include, but notfully comprise, chemotherapy, radiation, hormone therapy, differentimmunity-boosting medicines and procedures, hyperthermia and systemicmonoclonal antibody treatment. The latter can be labeled withradioactive elements, immunotoxins and chemotherapeutic drugs.

Radioactively labeled monoclonal antibodies were initially used withsuccess in lymphomas and leukemia, and recently in some carcinomas. Theconcept underlying the use of labeled antibodies is that the labeledantibody will specifically seek and bind to the carcinoma and, theradioactive element, through its decay, will irradiate the tumor insitu. Since radioactive rays travel some distance in tumors it is notnecessary that every carcinoma cell bind the labeled antibody. Thespecificity of the monoclonal antibodies will permit a selectivetreatment of the tumor while avoiding the irradiation of innocentby-stander normal tissues, that could be dose limiting. Chemotherapyproduces serious toxic effects on normal tissues, making thechemotherapy of carcinomas less than desirable, and the use ofradiolabeled monoclonal antibodies a valid alterative.

Non-human antibodies raised against human epitopes have been used forthe diagnosis and therapy of carcinomas as is known in the art. Alsoknown are the methods for preparing both polyclonal and monoclonalantibodies. Examples of the latter are BrE-2, BrE-3 and KC-4 (e.g. U.S.Pat. Nos. 5,077,220; 5,075,219 and 4,708,930.

The KC-4 murine monoclonal antibody is specific to a unique antigenicdeterminant, the “antigen”, and selectivity binds strongly to neoplasticcarcinoma cells and not to normal human tissue (U.S. Pat. No. 4,708,930to Coulter). The antigen appears in two forms in carcinoma cells, onlythe smaller of these forms being expressed in the cell membrane. Thelarger form appears only in the cytoplasm and has an approximate 490Kdalton molecular weight (range of 480,000-510,000). The second formoccurs at a higher density of expression, is found both in the cytoplasmand the membrane of carcinoma cells and has an approximate 438 Kdaltonmolecular weight (range of 390,000-450,000) as determined by gelelectrophoresis with marker proteins of known molecular weights. LabeledKC-4 was applied to the diagnosis and medical treatment of variouscarcinomas, particularly adenocarcinoma and squamous cell carcinomaregardless of the human organ site of origin.

The BrE-3 antibody (Peterson et al., Hybridoma 9:221 (1990); U.S. Pat.No 5,075,219) was shown to bind to the tandem repeat of the polypeptidecore of human breast epithelial mucin. When the mucin is deglycosylated,the presence of more tandem repeat epitopes is exposed and the bindingof the antibody increases. Thus, antibodies such as BrE-3 bindpreferentially to neoplastic carcinoma tumors because these express anunglycosylated form of the breast epithelial mucin that is not expressedin normal epithelial tissue. The preferential binding combined with anobserved low concentration of epitope for these antibodies in thecirculation of carcinoma patients, such as breast cancer patients, makesantibodies having specificity for a mucin epitope highly effective forcarcinoma radioimmunotherapy. A ⁹⁰Y-BrE-3 radioimmunoconjugate provedhighly effective against human breast carcinomas transplated into nudemice. Human clinical studies showed the ⁹⁰Y-BrE-3 radioimmunoconjugateto considerably reduce the size of breast tumor metastases without anyimmediate toxic side effects. Moreover, an ¹¹¹In-BrE-3radioimmunoconjugate was successfully used for imaging 15 breast cancerpatients, providing excellent tumor targeting in 13 out of 15 of thepatients. Out of all the breast tumor metastases occurring in anotherstudy, 86% were detected by ¹¹¹In-BrE-3. Unfortunately, 2 to 3 weeksafter treatment, the patients developed a strong human anti-murineantibody (HAMA) response that prevented further administration of theradioimmunoconjugate. The HAMA response, which is observed for numerousmurine monoclonal antibodies, precludes any long-term administration ofmurine antibodies to human patients. Similarly, other heterologousantibodies, when administered to humans, elicited similar antibodyresponses. The anti-heterologous human response is, thus, a substantiallimiting factor hindering the successful use of heterologous monoclonalantibodies as therapeutic agents, which could, otherwise, specificallyannihilate breast carcinomas, causing little or no damage to normaltissue and having no other toxic effects.

Chimeric antibodies are direct fusions between variable domains of onespecies and constant domains of another. Murine/human chimericantibodies prepared from other types of B cells binding to other typesof antigenic determinants have been shown to be less immunogenic inhumans than wide murine antibodies. These proved to be less immunogenicbut still in some cases an immune response is mounted to the rodentvariable region framework region (FR). A further reduction of the“foreign” nature of the chimeric antibodies was achieved by graftingonly the CDRs from a rodent monoclonial into a human supportingframework prior to its subsequent fusion with an appropriate constantdomain (European Patent Application, Publication No. 239,400 to Winter;Riechmann, et al., Nature 332:323-327 (1988)). However, the proceduresemployed to accomplish CDR-grafting often result in imperfectly“humanized” antibodies. That is to say, the resultant antibody losesaffinity (usually 2-3 fold, at best).

The ligand binding characteristics of an antibody combining site aredetermined primarily by the structure and relative disposition of theCDRs, although some neighboring residues also have been found to beinvolved in antigen binding (Davies, et al., Ann Rev. Biochem.59:439-473 (1990)).

The technologies of molecular biology have further expanded the utilityof many antibodies by allowing for the creation of class switchedmolecules whose functionality has been improved by the acquisition orloss of complement fixation. The size of the bioactive molecule may alsobe reduced so as to increase the tissue target availability of theantibody by either changing the class from the IgM to an IgG, or byremoving most of the heavy and light chain constant regions to form anF_(V) antibody. Common to all of these potentially therapeutic forms ofantibody are the required complementary determining regions (CDRs),which guide the molecule to its ligand, and the framework residues (FRs)which support the CDRs and dictate their disposition relative to oneanother. The crystallographic analysis of numerous antibody structuresrevealed that the antigen combining site is composed almost entirely ofthe CDR residues arranged in a limited number of loop motifs. Thenecessity of the CDRs to form these structures, combined with theappreciated hypervariability of their primary sequence, leads to a greatdiversity in the antigen combining site, but one which has a finitenumber of possibilities. Thus, its hypermutability and the limitedprimary sequence repertoire for each CDR would suggest that the CDRsderived for a given antigen from one species of animal would be the samederived from another species. Hence, they should be poorly immunogenic,if at all, when presented to a recipient organism.

Accordingly, there is still a need for a product of high affinity and/orspecificity for carcinoma antigens suitable for the detection andtherapy of carcinomas which elicits a lesser antibody response thanwhole non-human antibodies or chimeric antibodies containing, forinstance the entire non-human variable region.

SUMMARY OF THE INVENTION

This invention relates to a humanized mouse monoclonal antibody and itsglycosylated derivative which specifically and selectively bind to thehuman KC-4 antigen, the antibody consisting essentially of the variableregions of the light and heavy chains of the anti-KC-4 mouse antibodyhaving the ATCC No. HB 8710 or HB 8709, wherein specific amino acids inthe FR are substituted per chain with amino adds present in equivalentpositions in antibodies of other species, and the constant region of ahuman antibody.

Also provided are the corresponding DNA and RNA segments encoding themonoclonal antibody, a hybrid vector carrying the DNA, and a transfectedhost thereof.

Still part of this invention are in vitro methods of diagnosing cancerand for conducting immunohistochemistry assays of tissue slices, an exvivo method of purging neoplastic cells, and in vivo methods for imagingand therapy of cancer patients.

Other objects, advantages and features of the present invention willbecome apparent to those skilled in the art from the followingdiscussion.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

This invention arose from a desire by the inventors to improve onantibody technology suitable for use in diagnostic and therapeuticapplications, particularly for in vivo administration. The usefulmonoclonal antibodies obtained up to the present time have been preparedby fusing immortalized cell lines with B-cells of mouse or other animalorigin. However, in general, heterologous antibodies may only beadministered once to a human due to the detrimental immunologicaleffects they elicit. This is true for most heterologous antibodiesadministered For example, the repeated administration of murineantibodies to a subject elicits a strong human anti-murine antibody(HAMA) response, which precludes their further utilization astherapeutic agents in humans. These heterologous antibodies initiate animmediate adverse reaction in many human patients and are, thus,rendered inaffective for further administration as therapeutic agents.On the other hand, human monoclonal hybridoma cell lines have not yetbeen very stable and have, therefore, not been suitable for the largescale, repeated production of monoclonal antibodies.

The present inventors, thus, have undertaken the preparation ofanti-KC-4 humanized monoclonal antibodies maintaining the entire CDRs ofthe mouse antibodies of the same specificity and human constant regions,and substituting 7 amino acids in the heavy chain and 12 amino acids inthe light chain, the substituted amino acids being positioned in theframework regions (FRs) and being selected from those present inequivalent positions in other human antibodies, and the constant regionsof a human antibody. The hybridomas cells of the invention can producelarge quantities of monoclonal antibodies having a desirable highaffinity, specificity and selectivity for the human KC-4 antigen.

The present inventors have found, surprisingly, that these monoclonalantibodies substantially preserve the binding, specificity andselectivity of the whole corresponding mouse antibody while they areexpected to elicit a less detnmental detrimental immunological response.However, the simple preservation of the binding region of an antibodydoes not by itself ensure that the binding characteristics of theantibody will be maintained. Antibodies are glycopolypeptides that arefolded into specific conformations. When the glycoside portion of themolecule or portions of the amino acid sequence are perturbed orexcised, the folding pattern of the molecule may be perturbed. Thus, anydeletion or modification of the sequence of an antibody must be madetaking into consideration that its folding dependent properties may bediminished or even obliterated if the folding is substantially affected,even though the amino acid sequences involved in the binding of theantigen are preserved.

The present inventors selected the following strategy for thepreparation and manufacture of the antibodies of this invention. ThecDNAs that encode the variable chains of an antibody may be obtained byisolation of mRNAs from hybridoma cells and their mRNAs reverselytranscribed. The thus obtained cDNAs may be amplified by the polymerasechain reaction (PCR) and the DNAs obtained inserted into a vector, andoptionally sequenced and restriction enzyme cut. Thus, the cDNAsencoding the variable chain (F.sub.V) region of the light (V.sub.L) andheavy (V.sub.H) chains of an antibody having affinity and specificityfor the human KC-4 antigen may be reverse transcribed from the isolatedmRNAs. The variable region cDNAs may then be modified, with predesignedprimers used to PCR amplify them or synthesized de novo, cloned into avector optionally carrying DNA sequences encoding the human contrastregion(s), optionally sequenced, and then transfected into host cellsfor expression of the humanized anti-LC-4 anti-KC-4 antibodies. Thebinding specifications and binding constants of the humanized antibodiesmay then be determined and compared to those of the whole mouseantibodies.

X-ray crystallographic studies demonstrate that the framework structuresof the F.sub.V of different antibodies assume a canonical structureregardless of the species of origin, amino acid sequence, or ligandspecificity. This is generally taken as evidence that the ligand-bindingcharacteristics of an antibody combining site are determined primarilyby the structure and relative disposition of the CDRs, although someneighboring framework residues may also be involved in antigen-binding.Thus, if the fine specificity of an antibody is to be preserved, its CRDCDR structures, and parts of the neighboring residues, their interactionwith each other and with the rest of the valuable domains, must also bemaintained. These crystallographic studies point to the possible needfor retaining most, if not all, of the many interior and inter-domaincontact residues since the structural effects of replacing only a few ofthem cannot be predicted.

While at first the necessity of keeping these amino acids might seem todefeat the goal of decreasing immunogencity by “humanization”, theactual number of amino acids that must be retained has been determinedby the inventors to be small because of the striking similarity betweenhuman and murine variable regions. Moreover, many if not most, of theretained amino acids posses side chains that are not exposed on thesurface of the molecules and, therefore, may not contribute to itsantigenicity. Clearly, it is most of the exposed amino acids that aregood candidates for substitution since it is these amino acids that areexposed to the immunological environment of a mammal and may formepitopes of increased immunogenicity.

The challenge in humanizing the variable regions of the anti-KC-4 mouseantibody thus begins with the identification of the “important”heterologous amino acids. “Important” amino acids are defined herein asthose, for example, that are involved in antigen binding, contact theCDRs and the opposite chains, and have buried side chains. Ideally,these residues might be identified from a well characterizedthree-dimensional structure. However, when, as in the present case,direct structural data are not available, the inventors have,fortunately, made it possible to predict the location of these importantamino acids by analyzing other related antibody structures, especiallythose whose variable light and heavy regions belong to the same class.The classes of variable regions can be determined from their amino acidsequence.

One method by which these important amino acids may be identified hasbeen described for the case of the amino acids with buried side chainsby Padlan, E. A. (Padlan, E. A., “A Possible Procedure for Reducing theImmunogenicity of Antibody Variable Domains While Preserving TheirLigand-Binding Properties”, Molecular Immunology, 28:489-494 (1991)). Inthe present case, various antibody variable region structures werecompared using a computerized program that determines the solventaccessibility of the framework residues as well as their contacts withthe opposite domain as described by Padlan, E. A. (1991), supra.Surprisingly, a close examination of the fractional solventaccessibility reveals a very close similarity in the exposure patternsof the V_(H) and the V_(L) domains. Put in simple terms, regardless ofthe particular antibody in question, and of its amino acid sequence, theinventors have found that the buried residues occupy similar relativepositions in most antibodies.

A similar analysis can be done by computer modeling, to determine whichamino acids contact the CDRs and which contact the opposite domain. Atthis point, the Fab structures that are currently in the Protein Databank (Bernstein, F. C., et al., J. Mol. Biol. 112:535-542 (1977)) may beexamined to determine which FRs may be important in maintaining thestructure of the combining site. Thus, after a close inspection of manyhigh resolution three-dimensional structures of variable regions, thepositions of all important framework amino acids, that is, those thatcontact the CDRs, the opposite domain, and those whose side chains areinwardly pointed, may be tabulated. Keeping these amino acids, as wellas those from the CDRs, and finally those FR amino acids that may beinvolved in ligand binding, should insure to a great extent thepreservation of affinity. The precise identification of FR amino acidsthat are involved in ligand-binding cannot be generalized since itvaries for different antibodies. Nevertheless, conservative decisionscan be made to preserve the amino acids located in FRs that have a highprobability of contacting the antigen. These regions are generallylocated immediately adjacent to the CDRs and at the N-terminus of bothchains, because the surfaces of these regions are contiguous with theCDR surfaces.

Surprisingly, it is possible to keep all of these important amino acidsin a heterologous humanized antibody and still increase dramatically thesimilarity with a human consensus sequence. That is, the final number ofamino acids with murine identities differing from human identities thatare kept is typically small. This is possible because human frameworksthat are similar to the murine frameworks, especially at the positionsof the important amino acids, can be found. This is because many of theimportant amino acids have the same identities in both murine and humanantibodies.

All the amino acids that are determined to be not important by themethod described above may be replaced by their corresponding humancounterparts. The surface of the finally humanized antibody should lookvery much like that of a human antibody except for the antigen bindingsurfaces. The original shape of those binding surfaces, however, ismaintained by leaving the internal composition of the antibody intact,preserving inter-domain contacts and by keeping very few key amino acidsthat contact the CDRs.

a) Choosing the Best Human Framework to Use in the “Humanization” of anAntibody When its Structure is Known

At the present time, there are 11 Fab structures for which the atomiccoordinates are known and have been placed in the Protein Data Bank asshown in Table 1 below, 2 from human and 9 from murine antibodies.

TABLE 1 Fab Structures for Which Coordinates are in the Protein DataBank ANTIBODY RESOLUTION (A) R-VALUE PDB CODE HUMAN NEWM 2.0 0.46 3FABKOL 1.9 0.189 2FB4 MURINE McPC603 2.7 0.225 1MCP J539 1.95 0.194 2FBJHyHEL-5 2.54 0.245 2HFL HyHEL-10 3.0 0.24 3HFM R19.9 2.8 0.30 1F194-4-20 2.7 0.215 4FAB 36-71 1.85 0.248 6FAB B13I2 2.8 0.197 1IGF D1.32.5 0.184 1FDL

The contacts between side chains in the variable domains of the 11 Fabshave been collected and are presented in Tables 2 to 4 below. Theframework (FR) amino acids in the V_(L) domains that contact CDRs arelisted in Table 2 below.

TABLE 2 V_(L) Framework Residues That Contact CDR Residues in Fabs ofKnown Three-Dimensional Structure ANTIBODY POSITION J539 McPC603HyHEL-10 HyHEL-5 R19.9 4-4-20 36-71 B13I2 D1.3 NEWM KOL 1 GLU(2) ASP(5)ASP(10) ASP(3) ASP(8) ASP(4) ASP(11) 2 ILE(11) ILE(15) ILE(17) ILE(13)ILE(5) VAL(9) ILE(20) VAL(9) ILE(10) SER(3) 3 VAL(3) VAL(2) VAL(3)GLN(2) VAL(2) GLN(2) LEU(6) VAL(2) 4 LEU(7) MET(6) LEU(6) LEU(10) MET(9)MET(13) MET(7) MET(6) MET(7) LEU(4) LEU(6) 5 THR(1) THR(1) THR(2) THR(1)7 THR(4) 22 SER(6) 23 CYS(1) CYS(1) CYS(2) CYS(2) CYS(1) CYS(1) CYS(1)CYS(1) 35 TRP(3) TRP(2) TRP(4) TRP(2) TRP(5) TRP(4) TRP(4) TRP(1) TRP(2)36 TYR(12) TYR(16) TYR(8) TYR(10) TYR(22) TYR(13) TYR(15) TYR(8) TYR(14)TYR(13) TYR(11) 45 LYS(12) LYS(5) 46 PRO(3) LEU(6) LEU(4) ARG(15) LEU(5)VAL(14) LEU(5) LEU(10) LEU(6) LEU(2) LEU(6) 48 ILE(1) ILE(1) ILE(1)ILE(3) ILE(2) VAL(1) ILE(1) 49 TYR(28) TYR(29) LYS(13) TYR(12) TYR(40)TYR(22) TYR(22) TYR(16) TYR(25) TYR(25) 58 VAL(3) VAL(3) ILE(1) VAL(6)VAL(6) VAL(5) VAL(4) VAL(5) VAL(1) VAL(6) 60 ASP(1) ASP(2) ASP(4) ASP(2)62 PHE(1) PHE(1) PHE(1) 66 LYS(2) LYS(11) 67 SER(3) SER(1) 69 THR(3)THR(3) THR(5) THR(1) THR(4) THR(1) SER(1) 70 ASP(2) ASP(1) ASP(6) SER(2)71 TYR(14) PHE(23) PHE(17) TYR(17) TYR(24) PHE(1) TYR(17) PHE(19)TYR(16) ALA(3) ALA(4) 88 CYS(1) CYS(2) CYS(1) CYS(1) CYS(1) CYS(1)CYS(2) CYS(1) 98 PHE(8) PHE(8) PHE(10) PHE(5) PHE(8) PHE(4) PHE(8)PHE(14) PHE(14) PHE(3) PHE(7)

Those FR in the V_(H) domains that contact CDRs are listed in Table 3below.

TABLE 3 V_(H) Framework Residues That Contact CDR Residues in Fabs ofKnown Three-Dimensional Structure ANTIBODY POSITION J539 McPC603HyHEL-10 HyHEL-5 R19.9 4-4-20 36-71 913I2 D1.3 NEWM KOL 1 GLU(3) 2VAL(11) VAL(3) VAL(8) VAL(1) VAL(7) VAL(3) VAL(12) VAL(9) 4 LEU(2)LEU(5) LEU(5) LEU(2) LEU(1) LEU(1) LEU(1) LEU(1) LEU(1) 24 THR(2) VAL(6)ALA(1) 27 PHE(3) PHE(2) TYR(14) TYR(11) PHE(26) TYR(4) PHE(4) PHE(4)THR(1) PHE(3) 28 ASP(3) THR(5) THR(3) THR(6) THR(4) THR(2) THR(3) SER(1)ILE(2) 29 PHE(4) PHE(4) PHE(10) PHE(7) PHE(13) PHE(6) PHE(3) LEU(1)PHE(4) 30 THR(2) THR(6) SER(7) ASP(5) 36 TRP(2) 37 VAL(1) VAL(1) VAL(1)VAL(2) VAL(1) 38 ARG(1) ARG(2) ARG(4) LYS(2) LYS(1) ARG(4) LYS(2) ARG(1)ARG(3) 40 ARG(1) 46 GLU(3) GLU(4) GLU(1) GLU(2) GLU(3) GLU(4) GLU(9)GLU(1) GLU(1) 47 TRP(21) TRP(29) TYR(20) TRP(21) TRP(13) TRP(18) TRP(21)TRP(23) TRP(19) TRP(22) TRP(15) 48 ILE(1) ILE(1) MET(6) ILE(12) ILE(13)VAL(1) ILE(9) VAL(3) LEU(1) ILE(2) VAL(1) 49 ALA(2) ALA(2) ALA(2) ALA(2)66 ARG(11) ARG(3) ARG(2) ARG(2) ARG(1) 67 PHE(4) PHE(10) ILE(9) ALA(1)PHE(11) THR(5) PHE(12) LEU(6) VAL(2) PHE(10) 68 ILE(1) THR(1) THR(11)THR(2) 69 ILE(8) VAL(6) ILE(8) PHE(12) LEU(5) ILE(20) LEU(6) ILE(11)ILE(8) MET(4) ILE(9) 71 ARG(7) ARG(16) ARG(2) ALA(1) VAL(4) ARG(6)VAL(6) ARG(3) LYS(4) ARG(9) 73 ASN(1) THR(3) ASP(3) 76 LEU(4) LEU(7)TYR(9) ALA(1) ALA(1) VAL(2) ALA(1) LEU(6) VAL(4) PHE(5) LEU(5) 80 LEU(1)82 LEU(2) MET(1) LEU(1) 86 ASP(2) 92 CYS(1) CYS(1) CYS(1) 93 ALA(4)ALA(5) LEU(2) THR(3) ALA(1) THR(5) ALA(4) ALA(1) ALA(3) 94 ARG(38)ARG(24) ASN(11) HIS(2) ARG(30) ARG(23) ARG(14) ARG(30) ARG(22) ARG(27)103 TRP(5) TRP(9) TRP(2) TRP(2) TRP(5) TRP(2) TRP(4) TRP(4)

The FR amino acids, that contact the opposite domain and whichpresumably are the ones mainly responsible for the quaternary structureof the F_(V) domains are listed in Table 4 below.

TABLE 4 Framework Residues That Contact Framework Residues in theOpposite Domain in Fabs of Known Three-Dimensional Structure ANTIBODYPOSITION J539 McPC603 HyHEL-10 HyHEL-5 R19.9 4-4-20 36-71 913I2 D1.3NEWM KOL IN V_(L): 36 TYR(3) TYR(4) TYR(3) TYR(5) TYR(11) TYR(7) TYR(1)TYR(7) TYR(5) 38 GLY(10) GLN(4) GLN(9) GLN(5) GLN(5) GLN(3) GLN(6)GLN(12) GLN(6) GLN(7) GLN(8) 43 SER(7) PRO(1) SER(8) SER(5) THR(3)SER(3) SER(2) ALA(5) ALA(1) 44 PRO(10) PRO(14) PRO(8) PRO(11) PRO(7)ILE(20) PRO(16) PRO(16) PRO(7) PRO(13) 46 PRO(3) 85 MET(2) THR(5) VAL(1)ASP(12) 87 TYR(6) TYR(4) PHE(6) TYR(2) PHE(5) TYR(10) TYR(8) TYR(6)TYR(6) 98 PHE(11) PHE(8) PHE(7) PHE(12) PHE(12) PHE(12) PHE(8) PHE(13)PHE(12) PHE(10) PHE(15) 100  ALA(2) IN V_(B): 37 VAL(4) ILE(2) VAL(1)VAL(4) VAL(2) VAL(1) VAL(2) VAL(4) VAL(1) VAL(4) 39 GLN(10) GLN(4)LYS(8) GLN(5) GLN(5) GLN(3) GLN(6) GLN(10) GLN(6) GLN(4) GLN(7) 43ASN(4) GLN(7) LYS(6) ARG(19) 44 ARG(2) 45 LEU(13) LEU(12) LEU(6) LEU(14)LEU(8) LEU(11) LEU(13) LEU(14) LEU(11) LEU(16) 47 TRP(1) TYR(2) TRP(2)TRP(3) TRP(2) 91 TYR(6) TYR(4) TYR(3) TYR(8) PHE(3) TYR(2) PHE(4) TYR(3)TYR(5) TYR(3) 103  TRP(11) TRP(15) TRP(16) TRP(11) TRP(4) TRP(18)TRP(24) TRP(22) TRP(19) TRP(8) TRP(19) 105  GLN(5)

The buried, inward-pointing FR amino acids in the V_(L) domains, i.e.,those which are located in the domain interior, are listed in Table 5below.

TABLE 5 Inward-Pointing, Buried Framework Residues in the V_(I) of Fabsof Known Three-Dimensional Structure ANTIBODY POSITION J539 McPC603HyHEL-10 HyHEL-5 R19.9 4-4-20 36-71 B13I2 D1.3 NEWM KOL 2 ILE ILE ILEILE ILE VAL ILE VAL ILE 4 LEU MET LEU LEU MET MET MET MET MET LEU LEU 6GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN GLN 11 THR LEU LEU MET LEU LEULEU LEU LEU VAL ALA 13 ALA VAL VAL ALA ALA VAL ALA VAL ALA 19 VAL VALVAL VAL VAL ALA VAL ALA VAL VAL VAL 21 ILE MET LEU MET ILE ILE ILE ILEILE ILE ILE 23 CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS 35 TRP TRPTRP TRP TRP TRP TRP TRP TRP TRP TRP 37 GLN GLN GLN GLN GLN LEU GLN LEUGLN GLN GLN 47 TRP LEU LEU TRP LEU LEU LEU LEU LEU LEU LEU 48 ILE ILEILE ILE VAL ILE ILE ILE VAL ILE 49 PHE 58 VAL VAL ILE VAL VAL VAL VALVAL VAL VAL 61 ARG ARG ARG ARG ARG ARG ARG ARG ARG ARG 62 PHE PHE PHEPHE PHE PHE PHE PHE PHE PHE PHE 71 TYR PHE PHE TYR TYR PHE TYR PHE TYRALA ALA 73 LEU LEU LEU LEU LEU LEU LEU LEU LEU LEU LEU 75 ILE ILE ILEILE ILE ILE ILE ILE ILE ILE ILE 78 MET VAL VAL MET LEU VAL LEU VAL LEULEU LEU 82 ASP ASP ASP ASP ASP ASP ASP ASP ASP ASP ASP 83 PHE 84 ALA ALAALA ALA ALA ALA THR 86 TYR TYR TYR TYR TYR TYR TYR TYR TYR TYR TYR 88CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS 102 THR THR THR THR THR THRTHR THR THR THR THR 104 LEU LEU LEU LEU LEU LEU LEU LEU LEU LEU LEU 106LEU ILE ILE ILE ILE ILE VAL VAL

Those in the V_(H) domain are listed in Table 6 below.

TABLE 6 Inward-Pointing, Buried Framework Residues in the V_(H) of Fabsof Known Three-Dimensional Struture ANTIBODY POSITION J539 McPC603HyHEL-10 HyHEL-5 R19.9 4-4-20 36-71 B13I2 D1.3 NEWM KOL  2 VAL VAL VALVAL VAL VAL VAL  4 LEU LEU LEU LEU LEU LEU LEU LEU LEU LEU LEU  6 GLUGLU GLU GLN GLU GLU GLN GLU GLU GLN GLN  9 PRO 12 VAL VAL VAL MET VALVAL VAL VAL VAL VAL VAL 18 LEU LEU LEU VAL VAL MET VAL LEU LEU LEU LEU20 LEU LEU LEU ILE MET LEU MET LEU ILE LEU LEU 22 CYS CYS CYS CYS CYSCYS CYS CYS CYS CYS CYS 24 ALA THR VAL ALA ALA ALA ALA ALA VAL VAL SER27 PHE PHE ASP TYR TYR PHE TYR PHE PHE THR PHE 29 PHE PHE ILE PHE PHEPHE PHE PHE LEU PHE PHE 36 TRP TRP TRP TRP TRP TRP TRP TRP TRP TRP TRP38 ARG ARG ARG LYS LYS ARG LYS ARG ARG ARG ARG 40 SER 46 GLU GLU GLU 48ILE ILE MET ILE ILE VAL ILE VAL LEU ILE VAL 49 ALA ALA ALA ALA 66 LYSARG ARG LYS ARG ARG ARG ARG 67 PHE PHE ILE ALA THR PHE THR PHE LEU VALPHE 69 ILE VAL ILE PHE LEU ILE LEU ILE ILE MET ILE 71 ARG ARG ARG ALAVAL ARG VAL ARG LYS VAL ARG 76 SER 78 LEU LEU TYR ALA ALA VAL ALA LEUVAL PHE LEU 80 LEU LEU LEU MET MET LEU MET LEU LEU LEU LEU 82 MET METLEU LEU LEU MET LEU MET MET LEU MET   82C VAL LEU VAL LEU LEU LEU LEULEU LEU VAL LEU 86 ASP ASP ASP ASP ASP ASP ASP ASP ASP ASP ASP 88 ALAALA ALA ALA ALA ALA ALA ALA 90 TYR TYR TYR TYR TYR TYR TYR TYR TYR TYRTYR 92 CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS CYS 94 ARG ARG ASN HISARG ARG ARG ARG ARG ARG 107  THR THR THR THR THR THR THR THR SER THR109  VAL VAL VAL LEU LEU VAL LEU LEU LEU VAL VAL 111  VAL VAL VAL VALVAL VAL VAL VAL VAL VAL VAL

From the above, it may be seen that

(1) There are many FR amino acids that either contact the CDRs or theopposite domain, or are found in the domain interior.

(2) These FR amino acids, which could influence the structure of thecombining site, and thus the antigen-binding characteristics of anantibody, are different from antibody to antibody.

It is obvious from these results that no one structure can serve as theperfect and sole basis of all humanization protocols. In fact, to“humanize” the 9 murine antibodies shown Table 1 above the CDR-graftingwith a view to preserving their ligand-binding properties, the FR aminoacids listed in Table 2 to 6 above would have to be retained.

A search through the tables of immunoglobulin sequences (Kabet et al.,“Sequences of Proteins of Immunological lnterest”, 5th Ed., US Dept. ofHealth and Human Service, NIH Publication No. 91-3242 (1991)), showsthat human variable domain sequences are known that already have most ofthe FR amino acids that need to be preserved as shown in Table 7 below.

TABLE 7 Human Antibodies that are Most Similar in Sequence to MurineAntibodies of Known Three-Dimensional Structure MOST SIMILAR ANTIBODYDOMAIN HUMAN SEQUENCE HyHEL-10 VH 58P2′CL (77/112) VH FRAMEWORK 15P1′CL,ML1′CL (62/87) VH IMPT 58P2′CL, Ab26′CL, C6B2′CL (28/38) VL IARC/BL41′CL(73/107) VL FRAMEWORK IARC/BL41′CL (59/80) VL IMPT IARC/BL41′CL (30/37)HyHEL-5 VH ND′CL (74/116) VH FRAMEWORK 783c′CL, X17115′CL (63/87) VHIMPT 21/28′CL, 51P1′CL, 783c′CL, 8E10′CL, AND, KAS, NEI′CL, X17115′CL(25/37) VL HF2-1/17′CL, KAS (65/105) VL FRAMEWORK HF2-1/17′CL (57/80) VLIMPT BI, DEN, HF2-1/17′CL, KUE, REI, WALKER′CL, WIL (=) (27/36) R19.9 VH21/28′CL (73/119) VH FRAMEWORK 21/28′CL, 51P′CL, AND, LS2′CL, NEI′CL,(60/87) VH IMPT 21/28′CL, 8E10′CL, Ls2′CL (28/38) VL WALKER′CL (78/107)VL FRAMEWORK RZ (62/80) VL IMPT REI, WALKER′CL (33/36) 4-4-20 VH 30P1′CL(77/116) VH FRAMEWORK 2P1′CL, 3D6′CL (65/87) VH IMPT 4B4′CL, M26′CL(36/41) VL RPM1-6410′CL (91/112) VL FRAMEWORK GM-607-′CL (68/80) VL IMPTCUM, FR, NIM (33/26) J539 VH 30P1′CL, Vh38CL, 10′CL (81/118) VHFRAMEWORK 18/2′CL, 30P1′CL, M43 (71/87) VH IMPT 38P1′CL, 56P1′CL, M72,M74 (36/40) VL PA (62/105) VL FRAMEWORK LEN, WEA, (53/80) VL IMPT BI,DEN, KUE, REI, WALKER′CL, WIL (=) (26/35) McPC603 VH M72 (81/120) VHFRAMEWORK 4G12′CL, Ab18′CL, M72 (70/87) VH IMPT 56P1′CL, M72, M74,RF-SJ2′CL (36/42) VL FK-001′CL, LEN (91/113) VL FRAMEWORK LEN (70/80) VLIMPT LEN (38/42) 36-71 VH 21/28′CL (74/119) VH FRAMEWORK 21/28′CL,51P1′CL, 783c′CL, AND′CL, NEI′CL, X17115′CL, (61/87) VH IMPT 21/28′CL,8E10′CL (28/38) VL AG (76/105) VL FRAMEWORK RZ (63/80) VL IMPT REI, RZ,WALKER′CL (34/37)′ B13I2 VH 56P1′CL (83/119) VH FRAMEWORK 4B41CL,4G12′CL, M26′CL, M72, RF-SJ2′CL, Vh38Cl.10′CL (68/87) VH IMPT 56P1′CL,M72, M74, RF-SJ2′CL (37/39) VL RPM1-6410′CL (86/112) VL FRAMEWORKGM-607-′CL (69/80) VL IMPT CUM, NIM (36/39) D1.3 VH C6B2′CL (72/116) VHFRAMEWORK C6B2′CL (62/87) VH IMPT M60′CL (32/37) VL BR (75/107) VLFRAMEWORK HF2-1/17′CL (64/80) VL IMPT 3D6′CL, BI, DEN, EU, KUE, PA, REI,WALKER′CL, WIL (=) (32/36)

These human sequences are not necessarily those which are most similarto the murine antibodies, overall or in the framework regions only, butrather, those that possess the largest number of important amino acidsin common, the latter sequences being included in Table 7 above.

The number of murine amino acids that still need to be retained in orderto have all the important FR amino acids in the “humanized” versions ofthe murine antibodies as shown in Table 7 above, ranges from 21 (forHyHEL-5:12 in V_(H) and 9 in V_(L)) to 5 (for B1312:2 in V_(H) and 3 inV_(L)). These are not very many amino acids, considering that theresulting humanized molecules will probably retain most or all theirligand-binding characteristics. It is possible that there exist otherhuman sequences that are even more similar to these murine domains thatare not included in the compilation of Kabat, et al. (1991), supra. Whenmore sequences become available these may also be incorporated toimprove the pool of basic data available for use in the humanization ofantibodies.

b) Choosing the best human framework to use in the “humanization” of anantibody when its structure is not known.

In the absence of a three-dimensional structure, the identification ofthe FR amino acids that are crucial to maintain the combining sitestructure is not easily done. Nevertheless, some proposals may be madefrom the data shown in Table 2 to 6 above that have been collected inTables 8 and 9 below for the V_(L) and V_(H) domains.

TABLE 8 Framework Residues in V_(L) That Probably Need To Be Preservedin Order to Reproduce the Ligand Properties of the Original Antibody   CDR1 J539 EI.L.Q....T.A.....V.I.C sass-------svsslh WYQQ.....SP.PWIYMcPC603 DIVMTQ....L.V.....V.M.C rssgsllnsqnqknfla WYQQ.....PP.LLIYHyHEL-10 DIVL.Q....L.V.....V.L.C rasq------signnlh WYQQ.....SP.LLIKHyHEL-5 DIVL.Q....M.A.....V.M.C sass-------svnymy WYQQ.....SP.RWIY R19.9.IQMTQ....L.A.....V.I.C rasq------disnyln WYQQ.....T.KLLVY 4-4-20DVVMTO....L.V.....A.I.C rasq-slvbsqqntylr WYLQ.....PKVLIY 36-71DIQM.Q....L.A.....V.I.C rasq------dinnfln WYQQ.....I.LLIY B13I2.VLM.Qr...L.V.....A.ISC ranq-tillsdgdtyle WYLQ.....SP.LLIY D1.3DI.M.Q....L.A.....V.I.C rasq------nihnyla WYQQ.....SP.LLVY  CDR2  CDR3J539 eisklas .V..RF........Y.L.I..M...D.A.YYC qqwtyplit F...T.L.L.McPC603 gastres .V.DRF....S.TDF.L.I..V...D.A.YYC qndhsyplt F.A.T.L.I.HyHEL-10 yasqsis .I..RF......T.F.L.I..V...D..MYPC qqsnswpyt F...T.L.I.HyHEL-5 dtsklas .V..RF........Y.L.I..M...D.A.YYC qqwqr-npt F...T.L.I.R19.9 ytsrlhs .V..RF.......DY.L.I..L...D.ATY.C qqqsttprt F...T.L...4-4-20 kvsnrfs .V.DRF......T.F.L.I..V...D...Y.C sqsthvpwt F...T.L...36-71 ftsrsqs .V..RF......TDY.L.I..L...D.A.YFC cqqnalprt F...T.L.I.B13I2 kvsnrfs .V.DRF......T.F.L.I..Y...D..VYYC fqqshwppt F...T.L.I. D1.3ytttlsd .V..RF....S.T.Y.L.I..L...DF..YYC qbfwstprt F...T.L...

TABLE 9 Framework Residues in V_(H) That Probably Need to Be Preservedin Order to Reproduce the Ligand Properties of the Original AntibodyCDR1 J539 .V.L.E.....V.....L.L.C.A..FDF. kywms WVRQ.....LEWI. McPC603.V.L.E.....V.....L.L.C.T..FTF. dfyme WVRQ....RLEWIA HyHEL-10.V.L.E..P..V.....L.L.C.V..D.IT sdyws WIRK...N.LEYM. HyHEL-5...L.Q.....M.....V.I.C.A..YTF. dywis WVKWR....LEWI. R19.9.V.L.E.....V.....V.M.C.A..YTFT sygvn WVKW...W..EWI. 4-4-20...L.E.....V.....M.L.C.A..FTFS dyvan WVRQS....LEWVA 36-71EV.L.Q.....V.....V.M.C.A..YTF. sngin WVKQ.....LEWI. B13I2.V.L.E.....V.....L.L.C.A..FTF. rcams WVRQ...K.L.WVA D1.3.V.L.E.....V.....L.I.C.V..F.L. gygvm WVRQ.....LEWL.          CDR2 J539eihp--dsgtinytpslkd  KF.I.R.N....L.L.M..V...D.A.YYCAR McPC603asrnkgnkytteysasvkg  RFIV.R.T....L.L.M..L...D.A.YYCAR HyHEL-10yvs---ysgstyynpslks  RI.I.R......Y.L.L..V...D.A.YYC.N HyHEL-5eilp--gagstnyverfkg  KA.F.A......A.M.L..L...D...YYCLH R19.9yinp--gkgylsynekfkg  .TTL.V......A.M.L..L...D.A.YFC.R 4-4-20girnkpynystyysdsvkg  RFTI.R.D..S.V.L.M..L...D...YYCT. 36-71ynnp--gngyiaynekfkg  .T.L.V......A.M.L..L...D.A.YFCAR B13I2giss--ggsytyfpdtyag  RF.I.R......L.L.M..L...D.A.YYCTR D1.3miv---gdgntdynsalks  RL.I.K......V.L.M..L...D.A.YYCAR        CDR3 J539lhygyn-------sy  W.Q.T.V.V.. McPC603 nyygstwyf----dy  W...T.V.V..HyHEL-10 wdg----------dy  W...T.V.V.. HyHEL-5gnydf--------dg  W...T.L.V.. R19.9 sfyggsdlavyyfds  W...T.L.V.. 4-4-20syygm--------dy  W...T.V.V.. 36-71 seyyggsykf---dy  W...T.L.V.. B13I2yssdpfyf-----dy  W...T.L.V.. D1.3 erdyrl-------dy   W...T.L.V..

From Tables 8 and 9 above, it may be seen that many of the important FRamino acids flank the CDRs. Among these flanking positions are most ofthe FR amino acids that are involved in the contact with the oppositedomain as shown in Table 4 above, and many of those which are in contactwith the CDRs as shown in Tables 2 and 3 above. Moreover, almost all ofthe FR amino acids that have been observed to participate in the bindingto antigen (Amit, A. G. et al., Science 233:747-753 (1986); Sheriff, etal., P.N.A.S. (USA) 82:1104-1107 (1987); Padian, E. A., et al., P.N.A.S.(USA) 86:5938-5942 (1989); Tulip, et al., Cold Spring Harbor Symp.Quant. Biol. 54:257-263 (1989); Bentley, et al., Nature (London) 348:254-257 (1990)), are in these flanking regions. Thus, duringhumanization, not just the CDRs are retained, but also some of theresidues immediately adjacent to the CDRs. This provides a better chanceof retaining more of the ligand-binding properties of the originalantibody. The likelihood of retaining the antigen binding properties ofthe mouse antibody is even greater if the first few amino acids in theNH₂-termini of both chains are also retained, since some of them arefound to be in contact with CDRs as shown in Tables 2 to 3 above.Further, Tables 8 and 9 above also show many other framework positionsthat are deemed structurally important in all the case examined here.The murine residues at those positions should probably be retained iswell.

Alternatively, it may possible to reduce immunogenicity, whilepreserving antigen-binding properties, by simply replacing those exposedresidues in the framework regions which differ from those usually foundin human antibodies (Padlan, E. A. (1991), supra). This would humanizethe surface of the anti KC-4 murine antibody while retaining theinterior and contacting residues which influence is antigen-bindingcharacteristics. The judicious replacement of exterior residues shouldhave little, or no, effect on the interior of the domains, or on theinterdomain contacts. For example, the solvent accessibility patterns ofthe F_(V)S of J539, a murine IgA (κ) and of KOL, a human IgG1 (λ) havebeen found to be very similar (Padian, E. A. (1991), supra).

At the present, more than 35 different Fab structures have beenelucidated by X-ray diffraction analysis, although atomic coordinatesfor only 11 are currently in the Protein Data Bank as shown in Table 1above. Most of the available structures have been analysed to onlymedium resolution, some having been refined to only a limited extent.Eventually, atomic coordinates for more and better-refined structureswill become available, so that the “important” FRs will be more easilyassessed. This will improve the theoretical predictive record of thepresent method for determining the sequence of the humanized antibodies.

The design of the humanized anti-KC-4 murine antibody is reached instages as follows.

1—Choice of a murine model of known structure.

2—Choice of the human FR.

3—Identification of murine/human antibody differences.

4—Identification of important murine amino acids.

1) Choice of a xenogeneic model of known structure

The V_(H) and V_(L) domains of an antibody of desired specificity areclassified according to Kabat et al.(1991), supra. Then, another humanantibody may be chosen, whose structure has been determined, and whosevariable regions belong to the same classes and subclasses. Modeling themurine antibody in question to such structure ensures maximal chance forsuccess. This, however, is not absolutely necessary since the relativepositions of the important amino acids do not vary considerable even invariable regions of different classes. Thus, with less than a perfectmatch, this method has been applied to design the humanized anti-KC-4antibodies of this invention. Once the murine model is chosen, it may beapplied to identify the locations of important residues in the murineantibody to be humanized. Tables 2, 3, 4, 5, 6, 8 and 9 indicate thepositions of the important amino acids in several antibodies whosestructures have been determined to a high resolution level.

(2) Choice of the target species FR

The target species framework should, ideally, be consensus framework.That is, one that has a maximum number of amino acids in common with allhuman frameworks of the same class. This is important, because, the goalof humanization is to avoid an immunological response against theengineered humanized anti-KC-4 antibody.

The target species framework that is chosen is that which shares thegreatest number of important amino acids with the original murineantibody. Thus, in choosing the human FRs, the similarity between theimportant amino acids is more important that the overall similarity.

In practice, the sequences of the murine variable chains were alignedwith the consensus sequences from all variable region classes of theanti-KC-4 murine antibody and the number of differences in the aminoacids that must be retained from the murine antibody were scored. Thehuman consensus sequence(s) that score(s) the lowest number ofdifferences is (are) then chosen. These are the best antibodycandidates. Others with low numbers that are higher than the above mayalso be suitable, and are placed in a reserve pool, and so forth. Ifthere are too many differences in the chosen framework (e.g., more than16), then the same alignment procedure using all tabulated humansequences may be repeated in order to find a specific human frameworkwhose similarity with the murine sequence is maximized at the positionsof the important amino acids. Thus, most preferably, the target speciesFR should be a consensus sequence. Next preferable would be a frameworkof a common human antibody, and finally, the framework of any humanantibody.

(3) Identification of murine/human antibody differences

The murine sequences are then aligned with the human sequences and theposition of all amino acids that differ in the murine and in the humanframeworks are tabulated. Such a table contains the maximum number ofamino acids that can be changed towards the humanization of the antiKC-4 murine antibody (see, Table 19 below). If all those changes were tobe made, a so-called CDR-grafted antibody would be obtained. That is,only the original CDRs would be retained from the anti-KC-4 murineantibody. The affinity of a CDR-grafted antibody by itself would beconsiderably less than that of the original anti-KC-4 murine antibody.In order to maximize the chances for conserving the original affinity,the identities of all important amino acids must be preserved.

(4) Identification of important murine amino acids

In the first step towards humanizing an antibody, the amino acids thatare correspondingly important in the anti-KC-4 murine antibody chosen instep 1 are retained. In a subsequent step, however, the amino acids thathave been shown to occupy important positions in other murine antibodiesor in human antibodies may also be retained and are therefore taken outfrom the group of candidates to be mutated. The second step isparticularly appropriate if all chances that the amino acids in questioncould make contacts with the CDRs or with the opposite chains are to beavoided. Once the important murine amino acids are identified, the DNAsequence may be mutagenized to change all other amino acids, which forthe most part occupy exposed positions.

The present method was applied in the exemplary disclosure providedhereinbelow to the humanization of the anti-KC-4 murine antibodystarting from a chimeric antibody consisting of anti-KC-4 mouse variableregions and human constant regions. Murine and human antibodies, whosethree-dimensional structures have been deduced to a high degree ofresolution, were utilized as guidance in the choice of the amino acidsto be substituted in order to humanize this murine antibody. Informationon other murine antibodies from a Data Bank was used in the exemplarydisclosure provided below to modify the anti-KC-4 murine-human chimericantibodies with human amino acids.

The cDNAs encoding the anti-KC-4 humanized variable regions were thencloned into a vector containing sequences encoding constant regions of ahuman antibody placed under the same promoter. Although this is thecloning strategy utilized in the exemplary disclosure of this invention,other methods known in the art may also be utilized such asco-expression and the like. In the exemplary disclosure provided herein,the anti-KC-4 murine-human chimeric antibodies were considered byjoining the DNAs of the anti-KC-4 murine variable domain to a humanconstant domain (an effector agent) cloned into a hybrid vector, and theproduct expressed by transfecting the vector into myeloma cells. Thevariable regions of the chimeric antibodies were then modified at theDNA level to obtain the humanized chimeric antibodies. The modificationsto the variable regions of the peptides may either be conducted by PCRamplification with primers that are custom tailored to produce thedesired mutations, or by DNA synthesis.

The anti-KC-4 humanized antibodies exemplified below comprises thehumanized variable regions of the anti-KC-4 murine/human chimericantibody (U.S. Pat. No. 4,708,930 to Coulter discloses the anti-KC-4mouse antibody technology) and the kappa and gamma 1 constant region ofa human antibody. These human antibodies were characterized by theirmolecular weight and binding specificities, and shown to complete wellwith, or better than, the corresponding murine and chimeric antibodiesfor the KC-4 antigen. The humanized antibodies were shown to bind weaklyto normal breast, lung, colon and endometrium, and strongly to carcinomatissue sections by the ABC immunoperoxidase method. The portions of theCDR and FR regions of the non-modified peptides (murine F_(V) regions)and effector agents (human F_(C) regions) were shown in both cases to besubstantially identical to those of the murine and human antibodies fromwhich they were obtained. The anti-KC-4 humanized antibodies of thisinvention lacking any non-human constant region sequences possess lessforeign antigenic epitopes than the whole murine or chimeric antibodiesfrom which they were derived. Accordingly, they are expected to elicit aless complex immunogenic response in humans than the corresponding wholemurine antibodies and even than the murine/human chimeric antibodies.However, to what extent a portion of the murine FR amino acids may bereplaced without altering the binding characteristics of the CDRs couldnot have been predicted prior to this invention because of thesubstantial conformational alterations in the interior regions thataffect the binding of the CDRs to the antigen that may occur uponmodification of amino acid sequences.

Thus, the substantially pure, isolated anti-KC-4 humanized antibody ofthe invention specifically and selectively binds to the human KC-4antigen described in U.S. Pat. No. 4,708,930. The antibody consists of alight and a heavy chain consisting essentially of the variable region ofthe light and heavy chains of the anti-KC-4 murine antibody having theFRs substituted with seven amino acids for the light chain and twelveamino acids for the heavy chain present in equivalent positions inantibodies of other species, and the constant regions of a humanantibody.

The humanization procedure described here is designed to minimizepotential losses in antigen binding affinity that may result from theintroduced amino acids. In the case of the anti-KC-4 humanized antibodydescribed herein, seven amino acid changes were introduced in thevariable region of the light chain and twelve amino acid changes weremade in the variable region of the heavy chain. Furthermore, to minimizethe immunological response to the humanized antibody, target human aminoacid sequences were used that comprise the consensus sequences of allappropriate human variable regions. In one particularly preferredembodiment of the invention the anti-KC-4 humanized antibody consistsessentially of the amino acid sequence ID No. 50 of Table 24 and/or thesequence ID No. 51 of Table 25.

The present anti-KC-4 humanized monoclonal antibodies are providedeither as a naked peptide or in glycosylated form. When provided inglycosylated form, the antibodies are attached to a glycosyl residue(s)provided by the eukaryotic cell where it is expressed. When cloned andexpressed in a prokaryotic cell it is provided as the naked polypeptide,and the glycosyl residue(s) may be added thereafter, for example bymeans of glycosyl transferases as is known in the art.

The anti-KC-4 humanized antibodies of this invention may also be added aradioisotope by methods that are known in the art.

In a most preferred embodiment, the anti-KC-4 humanized antibodycomprises the humanized antibody expressed by the hybridoma cell linehaving the ATCC Accession No. HB 11,455 (HuKC-4V2), deposited under theBudapest Treaty on Sep. 23, 1993. This hybridoma cell line was depositedas the best mode of the invention known to the inventors.

The anti-KC-4 humanized antibodies of the invention are also provided asa composition along with a carrier or diluent for use in vitro,preferably a pharmaceutically-acceptable carrier or diluent for use invivo and ex vivo. The anti-KC-4 humanized antibody provided herein maybe present in the composition in an amount of about 0.001 to 99.99 wt %,more preferably about 0.01 to 20 wt %, and still more preferably about 1to 5 wt %. However, other amounts are also suitable. Carriers generally,and pharmaceutically-acceptable carriers in particular, are known in theart and need not be further described herein. The carrier may beprovided in a separate sterile container or in admixture with theantibody. Typically, saline, aqueous alcoholic solutions, albumin-salinesolutions, and propylene glycol solutions are suitable. However, othersmay also be utilized. When utilized for therapeutic purposes the proteicmaterial must be of a purity suitable for human administration, and thecomposition may contain other ingredients as is known in the art.Examples of these are other anti-neoplastic drugs such as adriamycin andmitomycin, cytoxan, PALA and/or methotrexate, among others. However,other therapeutic drugs, carriers or diluents, immunological adjuvantsand the like may be also be added. When the composition described aboveis utilized for in vivo imaging, it may comprise about 0.001 to 99.9 wt% humanize antibody, and more preferably about 0.01 to 25 wt % humanizedantibody. Typically, when the composition is utilized for therapeuticpurposes it may contain about 0.001 to 99.9 wt % humanized antibody, andmore preferably about 0.01 to 30 wt % humanized antibody. When utilizedfor the ex vivo purging of neoplastic cells from bodily fluids such asspinal fluid, the composition may comprise about 0.0001 to 50 wt %, andpreferably about 0.01 to 20 wt % humanized antibody. When applied to thein vitro diagnosis of carcinomas the composition of the invention maycomprise about 0.001 to 35 wt % humanized antibody, and more preferablyabout 0.01 to 10 wt % humanized antibody. Other amounts, however, arealso suitable.

Such products find one utility in the treatment of cancer, such asbreast, lung, ovary, endometrial, pancreas, prostate and colon cancers,among others. The anti-KC-4 humanized antibodies may be used for the invivo treatment of diagnosis of humans. The present analogue peptides areparticularly suitable for repeated administration to humans and for longterm therapy, such as is the case of metastases, and/or the reoccurrenceof tumors.

A kit for the diagnosis of cancer cells provided herein comprises theanti-KC-4 humanized antibody of the invention, and instructions for itsuse, and optionally a positive control, and heterologous immunoglobulinsselectively binding the constant regions of the antibody, protein G orprotein A. The diagnostic kit may also be provided with a radiosotope ora fluorescent label.

A cancer patient may be imaged in vivo and/or diagnosed byadministration of the anti-KC-4 humanized antibody of the invention inradiolabeled form, in an amount effective to reach the locus of thecancer and bind to the cancer cells, and further non-invasive detectionof any localized binding of the labeled anti-KC-4 humanized antibody tothe tumor cells. Typically, the anti-KC-4 humanized antibody may beadministered in an amount of about 0.001 to 5000 mg/kg weight pertreatment, more preferably about 0.01 to 5000 μg/kg weight pertreatment, and more preferably about 0.1 to 500 μg/kg weight pertreatment. However, other amounts may also be utilized. Radiolabels thatmay be utilized are ¹¹¹In, ¹²⁵I, ^(99m)Tc, and ¹³¹I, among others. Theseradiosotopes may be detected with a PET scanner, and with an NMR imagingand/or radioactivity counting apparatus that are in wide use by themedical community, depending on the radiolabel utilized.

A cancer may be diagnosed in vitro by contacting a biological samplewith the anti-KC-4 humanized antibody described here into form ananti-KC-4 humanized antibody-cancer cell antigen complex with any canceror cancer-associated cell antigen present in the sample, and detectingany complex formed. The biological sample is typically obtained from ahuman suspected of being afflicted with cancer. Suitable biologicalsamples are serum blood, sputum, feces, lymph fluid, spinal fluid, lungsecretions, and urine, among others. Clearly, any source of fluid,tissue and the like may be prepared for use in this method as is knownin the art.

The anti-KC-4 humanized antibody of this invention was shown to havetissue specificities similar to that of the anti-KC-4 murine antibody.The anti-KC-4 humanized monoclonal antibody was shown to bindspecifically and strongly to solid tumor tissue in the lung, colon,kidney, breast, stomach, prostate, pancreas, lymph node duct andlymphoma, and non-specifically and weakly to normal breast, kidney, andstomach tissue. The anti-KC-4 murine antibody also showed some weakbinding to normal tissue including spinal cord, uterus, thyroid, tongue,prostate, spleen, adrenal, lung, gall bladder, heart, lymph nodes,colon, liver, brain, testes, thymus, and placenta (U.S. Pat. No.4,708,930).

The present anti-KC-4 humanized antibodies are also applicable to thepurging of cancer cells from biological samples, be it fluid or tissuesamples. The purging of neoplastic cells from a fluid sample is part ofthe invention and may be practiced by contacting a biological fluidsuspected of comprising neoplastic cells with the anti-KC-4 humanizedantibody of the invention, and allowing the antibody to bind to anyKC-4-related antigen present on the cells, and separating the anti-KC-4humanized antibody cell complex from the remainder of the fluid.

This method may be utilized for purging unwanted cell ex vivo byextracting a biological sample from a patient, eliminating theneoplastic cells therefrom by separation of the anti-KC-4 humanizedantibody-cell complexes or by further addition of an effector such ascomplement or a toxin or a radioactive label that can act upon the celland then replenishing the purged sample to the patient. This istypically suitable for use with spinal taps where spinal fluid is rid ofcarcinoma cells prior to reinjection. Other fluids may also be treatedin this manner.

The present humanized antibodies may also be applied to thehistochemical assessment of the presence of cancer cells in a tissueobtained from a subject suspected of begin afflicted with cancer bymethods that are standard in the art, like the preparation of tissueslices and their fixation on a solid substrate to permit the applicationof the monoclonal antibody of the invention, and then the assessment ofany binding to neoplastic cells in the sample as indicated by theformation of complexes between the anti-KC-4 humanized antibody andantigens to which it selectively binds on the cells.

The growth or the size of a primary or metastasized cancer may beinhibited or reduced by administering to a subject in a need of thetreatment an effective amount of the anti-KC-4 humanized antibody of theinvention in radiolabeled form. Typically, the monoclonal antibodyprovided herein may be administered in an amount of about 0.001 to 2000mg/kg body weight per does, and more preferably about 0.01 to 500 mg/kgbody weight per dose. Repeated doses may be administered as prescribedby the treating physician. However, other amounts are also suitable.Generally, the administration of the antibody of the invention isconducted by infusion so that the amount of radiolabel present that mayproduce a detrimental effect may be kept under control by varying therate of administration. Typically, the infusion of one dose may last afew hours. However, also contemplated herein is the constant infusion ofa dose for therapeutic purposes that will permit the maintenance of aconstant level of the antibody of this invention in serum. The infusionof the monoclonal antibody of the invention may be conducted as follows.Intravenous (I.V.) tubing may be pretreated, e.g., with 0.9% NaCl and 5%human serum albumin and placed for intravenous administration. Theprescribed dose of the analogue peptide may be infused as follows.Unlabeled analogue peptide may be infused initially. 30 minutes aftercompletion of the unlabeled antibody infusion. ¹¹¹In-labeled and ⁹⁰Ylabeled antibody may be con-infused. The I.V. infusion may comprise atotal volume of 250 ml of 0.9% NaCl and 5% human serum albumin and beinfused over a period of about 2 hours depending on any rate-dependentside effects observed. Vital signs should be taken, e.g., every 15minutes, during the infusion and every one hour post infusion untilstable. A thorough cardiopulmonary physical examination may be doneprior to, and at the conclusion, of the infusion. Medications includingacetaminophen, diphenhydramine, epinephrine, and corticosteroids may bekept at hand for treatment of allergic reactions should they occur. Theadministration of the hybrid analogue peptide of practitioner.Typically, once a first dose has been administered and imaging indicatesthat there could be a reduction in the size of the tumor, whetherprimary of metastasized, repeated treatments may be administered everyabout 1 to 100 days, and more preferably every about 2 to 60 days. up toabout 2 years, and in some circumstances even for longer periods of timeor until complete disappearance of the tumor(s). The administration ofthe radiolabeled antibody of this invention is typically more useful fortherapeutic purposes when a primary tumor has, for example, beenexcised. Thus, it is primarily intended for “mopping-up” therapy aftersurgical intervention or for applications in cases of cancerousmetastases. It is in these cases that the present method is of greatestutility.

A pure, isolated polydeoxyribonucleotide that encodes the anti-KC-4humanized antibody of this invention may be applied to the preparationof the monoclonal antibody of this invention. In one preferredembodiment, the polydeoxyribonucleotide of the invention consistsessentially of a DNA sequence selected from the group consisting of DNAsequence ID Nos. 48 and/or 49 of Tables 21 and 22. These DNA sequencesmay be cloned for expression under the same promoter.

Also provided herein is a hybrid vector that comprises a vector carryingthe polydeoxyribonucleotide of this invention operatively linkedthereto. Typically, vectors capable of replication both in eukaryoticand prokaryotic cells are suitable. When the preparation a glycosylatedanalogue polypeptide is desired the vector should be suitable fortransfection of eukaryotic host cells.

This invention also encompasses a host cell that has been transfectedwith the hybrid vector described above. Suitable hosts are prokaryoticand eukaryotic hosts such as bacteria, yeast, and mammalian cells suchas insect cells and non-producing hybridoma cells, among others.Suitable vectors and/or plasmids for the transfection of each one ofthese types of hosts are known in the art and need not be furtherdescribed herein. Also known in the art are methods for cloning DNAsequences into each one of these types of vectors and for transfectingthe different types of host cells. Particularly preferred is the cellline having the ATCC Accession No. HB 11,455 (HuKC4V2).

Polyribonucleotides may be obtained by transcription of thepolydeoxyribonucleotides described above as is known in the art.Provided herein are polyribonucleotides consisting essentially ofoligoribonucleotides encoding the variable regions of the anti-KC-4humanized antibody and the constant regions of a human antibody. Thepolyribonucleotides may be prepared by cloning the desired DNA segmentsand then transcribing the thus obtained hybrid polydeoxyribonucleotideinto the corresponding RNA sequences.

The anti-KC-4 humanized antibody of the invention may be produced bycloning the polydeoxyribonucleotide encoding the antibody of theinvention into a vector to form a hybrid vector, transfecting a hostcell with the hybrid vector and allowing the expression of the anti-KC-4humanized antibody, and isolating the antibody from the cell culturemixture. The DNA segment encoding the anti-KC-1 humanized antibody maybe obtained by chemical synthesis or by the site-specific modificationof the DNA sequence encoding the variable region of the anti-KC-4 murineor murine-human chimeric antibody by PCR amplification with specificallydesigned primers as is known in the art. Preferably, the cloning andtransfection steps are conducted by cloning polydeoxyribonucleotidesencoding the variable region of the heavy or light chains of theanti-KC-4 murine antibody into a DNA segment carrying the genes for thehuman constant regions, and allowing the antibody chains to beexpressed. The expressed antibody chains may then be allowed to interactwith one another to form the double chain antibody modified as describedabove.

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples, which are includedherein for purposes of illustration only and are not intended to belimiting of the invention or any embodiment thereof, unless sospecified.

EXAMPLES Example 1 Methods Utilized

The procedures utilized herein for the reverse-transcription (RT) ofRNAs encoding the variable regions and the subsequent amplification ofthe cDNAs by the polymerase chain reaction (PCR) have been described(Orlandi, R., et al., “Cloning Immunoglobulin Variable Domains forExpression by the Polymerase Chain Reaction”, PNAS (USA) 86:3833-3837(1989); Coloma, M. J., et al., “Primer Design for the Cloning ofImmunoglobulin Heavy-Chain Leader-Fvs from Murine Hybridoma Cells Usingthe PCR”, Bio. Techniques 11:152-156 (1991); Gavilondo-Cowley, J. V., etal., “Specific Amplification of Rearranged Immunoglobulin Fv Genes fromMurine Hybridoma Cells”, Hybridoma 9:407-417 (1990)).

Total RNA is an adequate substrate for RT-PCR. Polyadenylated RNA wasutilized herein however, because it contains only minor levels ofcontaminating ribosomal RNA and practically no DNA. The polyadenylatedRNA was isolated with a Fast Track mRNA isolation kit (InvitrogenCorporation, Sand Diego, Calif.).

The oligonucleotides were synthesized on a PCR-Mate EP DNA synthesizermodel 391 (Applied Biosystems, Foster City, Calif.). A PCR murine Igprimer set was purchased from Novagen (Madison, Wis.), and complementaryDNA (cDNA) was prepared with an RNA PCR kit (Perkin Elmer-Cetus,Norwalk, Conn.).

PCR DNA fragments were cloned directly into pCR1000, using a TA cloningkit (Invitrogen Corporation, San Diego, Calif.) Plasmid DNA was isolatedwith a kit purchased from Qiagen (Tchapsworth, Calif.), and DNAsequencing was conducted with a Sequenase 2.0 DNA sequencing kit (UnitedStates Biochemical, Cleveland, Ohio) using aqueous 5′α-³⁵SdATP at 600mCi/mmol (Amersham Corporation, Arlington Heights, Ill.).

Sequence analyses were performed on a Macintosh computer using theprogram GeneWorks (IntelliGenetics, Inc. Mountain View, Calif.).

Example 2 PCR Primers used in First Isolation of Anti-KC-4 cDNAs

The PCR primers were purchased from Novagen (Madison, Wis.). Theirsequences, reproduced from the booklet provided by Novagen, are shown inTable 10 below.

TABLE 10 PCR Primer Sequences MulgκV_(L)5′-C: sense primer mix for kappaleader. ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCTG (Seq. ID No: 1)ACTAGTCGACATGGAGWCAGACACACTCCTGYTATGGGT (Seq. ID No: 2)ACTAGTCGACATGGATTTWCAGGTGCAGATTWTCAGCTTC (Seq. ID No: 3) MulgκV_(L)3′-1:antisense kappa constant region. CCCAAGCTTACTGGATGGTGGGAAGATGGA (Seq. IDNo: 4) MulgV_(H)5′-F: sense primer mix for heavy chain leader.ACTAGTCGACATGRACTTTGGGYTCAGCTTGRTTT (Seq. ID No: 5)ACTAGTCGACATGAGAGTGCTGATTCTTTTGTG (Seq. ID No: 6)ACTAGTCGACATGGATTTTGGGCTGATTTTTTTTATTG (Seq. ID No: 7) MulgγV_(H)3′-2:antisense gamma constant region. CCCAAGCTTCCAGGGRCCARKGGATARACIGRTGG(Seq. ID No: 8)

Example 3 Amplification of cDNAs Encoding anti-KC-4 Antibody F_(V)Regions

The cDNAs that encode the anti-KC-4 murine immunoglobulin V_(H) andV_(L) were prepared by PCR from polyadenylated RNA isolated from 100million KC-4 hybridoma cells. All clones were obtained from independentPCRs. The sequences of the primers are given in Example 2 above. Primersare specific for either the leader peptide region or for the constantregions. The primer combinations utilized herein are shown in Table 11below.

TABLE 11 Primer Combination for PCR Amplifications Clone No. Primercombinations V_(L)  96 MulgκV_(L)5′-C + MulgκV_(L)3′-1 107MulgκV_(L)5′-C + MulgκV_(L)3′-1 K1 JO20 + JO21 V_(H)  66 MulgV_(H)5′-F +MulgγV_(H)3′-2 209 MulgV_(H)5′-F + MulgγV_(H)3′-1 H3 JO22 + JO24 H7JO22 + JO24

Example 4 Isolation of Amplified anti-KC-4 V_(L) and V_(H) cDNA andSequences

The PCR products were cloned, without prior purification, into pCR1000(Invitrogen) and sequenced in both directions. The V_(H) and V_(L) DNAsequences and their derived protein sequences are shown in Tables 12,13, 14, and 15 below.

TABLE 12 V_(L) Nucleotide sequences anti-KC-4 V_(L) (kII-Jk2) ATG AAGTTG CCT GTT AGG CTG TTG GTG CTG ATG TTC TGG ATT CCT GCT TCC AGC AGT GATGTT TTG ATG ACC CAA ACT CCT CTC TCC CTG CCT GTC AGT CTT GGA GAT CAA GCCTCC ATC TCT TGC AGA TCT AGT CAG AGC ATT GTA CAT AGT AAT GGA AAC ACC TATTTA GAA TGG TAC CTG CAG AAA CCA GGC CAG TCT CCA AAG CTC CTG ATC TAC AAAGTT TCC ATC CGA TTT TCT GGG GTC CCA GAC AGG TTC AGT GGC AGT GGA TCA GGGACA GAT TTC ACA CTC AAT ATC AGC AGA GTG GAG GCT GAG GAT CTG GGA ATT TATTAC TGC TTT CAA GGT TCA CAT GTT CCG TAC ACG TTC GGA GGG GGG ACC AAG CTGGAA ATA AAA C (Seq. ID No: 13)

TABLE 13 V_(H) Nucleotide sequences anti-KC-4 V_(H) (IIID-D9-JH3) ATGGAC TTT GGG CTC AGC TTG GTT TTC CTT GTC CTT ATT TTA AAA GGT GTC CAG TGTGAA GTG CAG ATG GTG GAG TCT GGG GGA GTG AAG CCT GGA GGG TCC CTG AAA CTCTCC TGT GCA GCC TCT GGA TTC GCT TTC AGT AGC TAT GCC ATG TCT TGG GTT CGCCAG GAG AAG AGG CTG GAG TGG GTC GCA GAA ATT AGT AGT GGT GGT AAT TAC GCCTAC TAT CAA GAC ACT GTG ACG GGC CGA TTC ACC AGA GAC AAT GCC AAG AAC ACCCTG TAC CTG GAA ATG AGC AGT CTG AGG TCT GAG GAC ACG GCC ATG TAT TAC TGTGCA AGG GAG GGT ATC CCG GCC TGG TTT GCT TAC TGG GGC CAA GGG ACT CTG GTCTCT GTC TCT GAC G (Seq. ID No: 14)

These cDNA sequences are accurate since in both cases they wereidentical for clones that were prepared from independent reversetranscription reactions. The derived protein sequences are shown inTables 14 and 15 below.

TABLE 14 V_(L) anti-KC-4 Amino Acid Sequences (kII-Jk2)MKLPVRLLVLMFWIPASSS (Seq. ID No: 15) FR1 DVLMTQTPLSLPVSLGDQASISC (Seq.ID No: 16) CDR1 RSSQSIVHSNGNTYLE (Seq. ID No: 17) FR2 WYLQKPGQSPKLLIY(Seq. ID No: 18) CDR2 KVSIRFS (Seq. ID No: 19) FR3GVPDRFSGSGSGTDFTLNISRVEAEDLGIYYC (Seq. ID No: 20) CDR3 FQGSHVPYT (Seq.ID No: 21) FR4 FGGGTKLEIK (Seq. ID No. 22)

TABLE 15 Anti-KC-4 V_(H) Amino Acid Sequences (IIID-D9-JH3)MDFGLSLVFLVLILKGVQC (Seq. ID No: 23) FR1 EVQMVESGGGLVKPGGSLKLSCAASGFAFS(Seq. ID No: 24) CDR1 SYAMS (Seq. ID No: 25) FR2 WVRQSPEKRLEWVA (Seq. IDNo: 26) CDR2 EISSGGNYAYYQDTVTG (Seq. ID No: 27) FR3RFTISRDNAKNTLYLEMSSLRSEDTAMYYCAR (Seq. ID No: 28) CDR3 EGIPAWFAY (Seq.ID No: 29) FR4 WGQGTLVSVSA (Seq. ID No: 30)

The sequences were interpreted as described by Kabat et al. (1991),supra. The residues that are underlined in the protein sequencescorrespond to PCR primer. The mature V_(L) and V_(H) chains begin atamino-acids D and E of framework 1 (FR1), respectively.

Framework and CDR protein segments were identified according to Kabat etal. (1991), supra. V_(L) is a group II κ chain. Part of the CDR 3 andall of the framework 4 (FR4) are encoded by Jk2. V_(H) belongs to groupIIId. CDR 3 and FR 4 resulted from a genomic recombination involvingminigenes D9 and JH3. There is an asparagine glycosylation site in thelight chain in FR3. The site reads NIS (Asn IIe Ser).

Example 5 Comparison of cDNA-deduced Amino Acid Sequence with DirectlyDetermined N-Terminal Fragment Sequence

A comparsion between the cDNA-derived polypeptide sequence and the aminoacid sequence determined directly on the purified anti-KC-4 monoclonalantibody was undertaken. The results are shown in Table 16 below.

TABLE 16 Comparison of cDNA-deduced with Directly Determined N-TerminalAmino Acid Sequences FIRST BAND TOP V_(H), cDNA-deducedEVQMVESGGGLVKPGGSLKLS (Seq. ID No: 31) V_(H), Protein sequenceEVQMVESGGGLVKPGGXLKLS (Seq. ID No: 32) SECOND BAND V_(L,) cDNA-deducedDVLMTQTPLSLPVSLGDQASI (Seq. ID No: 33) V_(L), Protein sequenceDVLMTQTPLSLPVXXGDQASI (Seq. ID No: 34) THIRD BAND V_(L), cDNA-deducedDVLMTQTPLSLPVSLGDQASI (Seq. ID No: 35) V_(L), Protein sequenceDVLMTQTPLSLPVSLGDQASI (Seq. ID No: 36) X uncertain or alternative calls.

A sample of anti-KC-4 chimeric antibody (approximately 190 μg) wasreduced with 5% beta-mercaptoethanol (65° C. for 15 min.), separated onthree lanes of a 10% SDS polyacrylamide gel, and electroblotted onto aProBlott membrane (Applied Biosystems, Foster City, Calif.) in 90% 30 mMCAPS pH11, 10% methanol, for 1 hour at 25 V and at 4° C. The transferredprotein species were stained with Commassie Brillant Blue. 3 bands wereseen in each lane, of which 2 migrated as expected for a heavy and lightchain. The third band migrated above the light chain. Amino acidsequencing was performed directly on the immobilized bands by theBiotechnology Instrumentation Facility, University of California,Riverside. The amino acid sequence given here is the sequencer's bestguess.

The close match betweenther deduced amino acid sequence and the directlydetermined amino terminal sequence indicates that the cloned cDNAsencode the authentic anti-KC-4KC-4 F_(V) region.

Example 6 Construction of Vectors Expressing Murine-Human Chimericanti-KC-4 Antibody

The two expression vectors pAG4622 and pAH4604 described in Coloma etal. (Coloma, M. J., et al., “Novel Vectors for the Expression ofAntibody Molecules Using Variable Regions Generated by PCR”, J. Immunol.Methods 152:89-104 (1992)). These were kindly provided by S. L. Morrison(Dept. of Mircobiology and Molecular Genetics, UCLA), U.S. applicationSer. No. 07/798,696; PCT/US91/10207. The construction and expression ofchimeric genes were performed as described by Coloma et al., supra.

Oligonucleotides synthesized and used in a PCR to produce V_(H) andV_(L) fragments with the correct ends for insertion into the pAG4622 andpAH4604 expression vectors are shown in Table 17 below.

TABLE 17 PCR Primers Sequences JO20 - sense kappa leader GGG GATATC CACCATG AAG TTG CCT GTT AGG CTG TTG (Seq. ID No: 9) JO21 - antisense JK2 CCCGTCGACTTAC G TTT TAT TTC CAG CTT GGT CCC CCC T (Seq. ID No: 10) JO22 -sense V_(H) leader GGG GATATC CACC ATG GAC TTT GGG CTC AGC TTG GTT TT(Seq. ID No: 11) JO24 - antisense JH3 CCC GCTAGC TGC AGA GAC AGA GAC CAGAGT CC (Seq. ID No: 12)

The original pCR1000 clones were the starting templates for the PCR. Thenew PCR products were cloned back into PCR1000 and their sequenceconfirmed. Correctly modified and amplified fragments were excised witheither EcoR V and Sal I (for V_(L)) or with EcoR V and Nhe I (forV_(H)). These fragments were then ligated into the respective vectors,which had been cut open with the appropriate restriction enzymes. Boththe vectors and the inserts were purified from an agarose gel prior toligation, using the Bio101 GeneClean kit (glass beads) (La Jolla,Calif.).

Example 7 Expression of the anti-KC-4 Chimeric Antibody Gene

Once inserted in pAG4622 and pAG4604, the V_(H) and V_(L) encodingregions in the anti-KC-4 murine-human chimeric antibody constructs weresequenced once again to verify their accuracy. The transfection of thenon-producer myelmoa cell line SP2/0-Ag14, (ATCC No. CRL 1581) andisolation of polypeptide was conducted as described in Coloma et al.,(1992), supra

Example 8 Production of Chimeric Antibody in Transfected Hosts

After ten days, stable transfectant colonies were clearly established ata frequency of approximately 1/10,000. Transfected cells were culturedeither in Dulbecco's modified Eagle's medium (DME): fetal bovine serum(FBS), 90:10 (v/v) or in a mixture of DME:RPMI:FBS, 45:45:10 (v/v/v) orRPMI:FBS, 90:10 (v/v). Penicillin and streptomycin were added to preventbacterial growth. Histidinol was added to the medium, at 5 mM, in orderto select for transfections. The colonies were transferred to normalmedium (without histidinol) and the supernatants from stabletransfectants were assayed for the presence of the murine-human chimericanti-KC-4 antibody. This was done by capturing the secreted murine-humanchimeric anti-KC-4 antibody with a plate-bound goat anti-human-κantibody and developing with goat anti-human-γ antibody as described byColoma et al. with the following modification. The secondary antibodyutilized herein was radiolabeled with ¹²⁴I.

Example 9 Confirmation of anti-KC-4 Murine Human Chimeric AntibodyExpression

The supernatants were assayed for binding to human milk fat globule(HMFG) as described by Ceriani et al. (Ceriani R. L., et al., DiagnosticAbility of Different Human Milk Fat Globule Antigens in Breast Cancer”,Breast Cancer Res. Treat. 15:161-174 (1990)). HMFG was bound to themicro-titer plates as described previously (Certiani R. L., “Solid PhaseIdentification and Molecular Weight Determination of Cell MembraneAntigens with Monoclonal Antibodies”, in: Monoclonal antibodies andfunctional cell lines. Progress and application, Bechtol, K. B., McKem,T. J., and Kennett, R., Eds., Plenum Press, New York, pp 398-402(1984)). The bound anti-KC-4 chimeric antibody (to kappa chainpolyclonal antibodies HMFG) was detected with either goat anti-humangamma chain or goat anti-human kappa chain polyclonal antibodiesconjugated to ¹²⁵⁻I. Most colony supernatants were positive by bothassays. The colonies that secreted the highest level of chimericantibody in the supernatants, as determined by these assays, weresubcloned.

Example 10 Western Blot

75 μl of the culture supernatant was added to 20 μl of 4× Laemmli bufferand 5 μl β-mercaptoethanol and the mixture was heated at 65° C. for 15min., in order to reduce antibody disulfide bonds and, thus, separateheavy from light chains 20 μl of the treated sample was chromatographedin duplicate lanes on a 10% SDS polyacrylamide gel together with otherantibodies that were treated similarly and that were loaded forcomparison. Pre-stained size markers (BioRad, Richmond, Calif.) werealso loaded. The chromatographed proteins were electroblotted onto aProBlott membrane (Applied Biosystems, Foster City, Calif.) in 90% 30 mMCAPS pH11, 10% methanol, for 1 hour at 25 V and at 4° c. The membranewas cut into 2 parts containing identical antibody samples. The 2membranes were immersed in 20% bovine calf serum in PBS and shakenslowly at room temperature for 1 hour 35 min. ¹²⁵I-labeled goatanti-human κ chain antibody was added to one membrane and ¹²⁵I labeledgoat anti-human γ chain antibody to the other membrane. Antibodies werelabeled at a specific activity of approximately 10 mCi/mg using thechloramine T method as described by Ceriani, R. L. and Blank, E. W.(1988), the labeled antibodies were diluted to 4,000 cpm/μl in RIAbuffer.

After incubating 3 hours at room temperature the blots were washed twicein TBS for 10 min each time, once in TBST (50 mM TRIS pH7.5, 3 mM EDTA25 mM NaCl) 10 min and once more in TBS (TBS with 0.05% Tween 20) for 10min. The membranes were dried and exposed to Kodak XAR film.

-   -   Western blot analysis of culture supernatants revealed that        three antibody chains were expressed that correspond to the        three antibody chains seen in the original anti-KC-4 murine        antibody. These were a heavy chain that stained with goat        anti-human γ chain ¹²⁵I-labeled antibody, and two light chains        that stained with goat anti-human κ chain ¹²⁵I-labeled antibody        (Figure not shown).

The treatment of the original anti-KC-4 murine antibody withN-glycosidase F (Boehringer Mannheim GmbH Germany) following therecommendations of the manufacturer, produced a noticeable decrease inthe intensity of the “top” chain and a concomitant increase in theintensity of the bottom light chain (FIG. not shown).

The explanation for the existence of an extra light chain is that thischain is glycosylated. Three lines of evidence substantiate this. First,the detection of an asparagine-linked glycosylation site in the aminoacid sequence of the light chain. That is the triad NIS (Asn-Ile-Ser) inframework 3. Second, the decrease of the intensity in the putativeglycosylated band after treatment with N-glycosidase F, whileconcomitantly the intensity of the non-glycosylated band was increased.Finally, 2 corresponding light chain bands are seen in the chimerciantibody version.

The extra light chain in the chimeric version cannot be a contaminantsince it was specifically stained by a goat anti-human κ chain antibody.It can only be a product expressed by pAG4622. Thus both light chainsmust have the same V_(L) amino acid sequence and the same human constantregion. These observations show that approximately half of the lightchains of both the anti-KC-4 murine and chimeric antibodies areglycosylated at the asparagine-linked glycosylation site.

Example 11 Tissue Binding Studies

The supernatants from stable transfectants were assayed for the presenceof the anti-KC-4 murine-human chimeric antibody as described using thevectastain ABC method (Vector Labs, Burlingame, Calif.).

The chimeric antibody secreted in the supernatant bound both HMFG andBEM very strong. In addition, the supernatants containing anti-KC-4murine-human chimeric antibody were used to stain human breast carcinomatissue sections by using the immunoperoxidase immunohistochemicalstaining technique. The intensity of the staining was comparable to thatobtained with the original murine monoclonal antibody.

The anti-KC-4 monoclonal antibody is known to bind the human milk fatglobule and the breast epitherlial mucin. This binding specificity ofthe anti-KC-4 murine monoclonal antibody was maintained even after therecombinant procedure. The anti-KC-4 chimeric antibody bound verystrongly to HMFG and BEM as determined by a radioassay (Ceriani, et al.,Breast Cancer Res. Trent. 15:161 (1990)). In addition, the anti-KC-4chimeric antibody bound several human beast tumors in histopathologicalsections in a manner comparable to the anti-KC-4 murine monoclonalantibody, detected by immunostaining using the vectastain ABC method(supra). This specificity of binding demonstrated the retained bindingreactivity of the variable regions of anti-KC-4 murine antibody by thepolypeptide of the invention when attached to the human F_(C) fragment.

Example 12 Approach for Humanization of Antibodies

The present humanization approach is based on Padlan, E. A., “Choosingthe Best Framework to Use in the Humanization of an Antibody byCDR-Grafting: Suggestions from 3-D Structural Data”, AntibodyEngineering 2nd. Annual Conf. San Diego, Calif. (Dec. 16-17, 1991).

The fine specificity may be preserved in a “humanized” antibody only ifthe CDR structures, their interaction with each other, and theirinteraction with the rest of the variable domains can be maintained.(Padlan, E. A. (1991) supra). This requires the preservation of residuesof the FR amino acids which contact the CDRs, those which are involvedin the V_(L)-V_(H) contact, and those which are buried and couldinfluence the overall domain structure and the structure of thecombining site.

By examination of murine Fab structures, for which atomic coordinatesare available, the FR amino acids that are probably “important” inmaintaining the structure of the combining site may be determined(Padlan, E. A., 8th International Congress of Immunol., Budapest,Hungary, Abstracts p. 19 (August 2-28, 1992)).

The specificity of an antibody depends on the CDR structures andsometimes, on some of its neighboring residues as well. These CDRstructures, in turn depend on contacts with framework amino acids and onthe interaction of the V_(L) and V_(H) domains. Thus, to ensure theretention of binding affinity, not only the CDR residues must bepreserved, but also those FR residues that contact either the CDRs ortheir opposite domains, as well as all buried residues, which give shapeto the variable domains. The buried amino acids are placed in exactlythe same positions in human and in murine frameworks (Padlan, E. A., “APossible Procedure for Reducing the Immunogenicity of Antibody VariableDomains While Preserving Their Ligand-Binding Properties”, MolecularImmunology 28:489-498 (1991)).

This approach was applied to design humanized analogues of the variableregions of the murine antibodies of the invention. The humanization ordesign of the exemplary analogue peptide provided herein was undertakenas follows. The identification of the residues, which are most probably“important” in preserving the combining site structure, permits theselection of the best human FR sequences to use in the “humanization” ofeach chimeric antibody of known structure or analogue peptides of theinvention. The results of the analysis can be used also to predict whichFR amino acids should probably be retained in those cases where nothree-dimensional structural data are available.

The present procedure was designed to reduce the immunogenicity of thexenogeneic antibodies by preparation of their chimeric derivatives orfragments thereof while preserving their antigen-binding properties. Ingeneral, the antigen binding properties of an antibody are primarilydetermined by its CDRs. The CDRs of the murine antibody were therefore,completely retained. In addition, the FR amino acids in the murineantibody, that are judged as probably important in maintaining thecombining site structure, were also retained in the humanized molecule.The remainder FR amino acids were changed to match those of the chosenhuman FR.

Example 13 Choice of Murine Model of Known Structure for Humanization ofanti-KC-4 Antibody

The classification of the V_(H) and V_(L) domains of an antibody such asthe anti-KC-4 antibody was done according to Kabat et al. (Kabat, E. A.,et al., “Sequence of Proteins of Immunological Interest” NIH (1991). TheKC-4G3 kappa chain V_(L) domain belongs to group II and the V_(H) domainbelongs to group IIId. A murine antibody was then found, whose structurehad been determined, and whose variable regions belong to the sameclasses. The anti-myobemerythrin peptide antibody B1312 fits theserequirements since, like the anti-KC-4 murine antibody, it has V_(L) andV_(H) domains belonging to groups II and IIId (Stanfield, R. L. et al.,“Crystal Structures of an Antibody to a Peptide and its complex withPeptide Antigen at 2.8 Å”, Science 248:712-719 (1990)). Thus, thethree-dimensional structures of antibodies the anti-KC-4 and B1312antibodies should be similar, and the humanization of the anti-KC-4antibody may be modeled after B1312.

Example 14 Choice of Target Human Framework for Humanization of Chimericanti-KC-4 Antibody

The choice of the target human framework was based strictly on thesimilarity at the residues that were judged to be structurally importantaccording to the B1312 model. That is, only amino acids that could beinvolved in contacts with CDRs of the opposite chain, or amino acidswhose side-chains were predicted to be inwardly pointed. The positionsof these amino acids are shown in Table 18 below.

TABLE 18 Important Amino Acid Positions for anti-KC-4 Antibody LightChain Variable Region Framework 2, 3, 4, 6, 7, 11, 13, 19, 21, 22, 23,35, 36, 37, 38, 43, 44, 46, 47, 48, 49, 58, 60, 61, 62, 69, 71, 73, 75,78, 82, 85, 86, 87, 88, 98, 102, 104 and 106. Heavy Chain VariableRegion Framework 2, 4, 6, 12, 18, 20, 22, 24, 27, 28, 29, 36, 37, 38,39, 43, 45, 46, 47, 48, 49, 66, 67, 69, 71, 78, 80, 82, 82c, 86, 88, 90,91, 92, 93, 94, 103, 107, 109 and 111.

The numbering system is conventionally accepted (Kabat, et al. (1991),supra) and is shown in Tables 10 and 11 above. In this case, theconsensus sequences of all human F_(V) regions were selected as thetarget human framework to minimize the immunogenicity of the product.

First, the sequences of the murine variable chains were aligned withconsensus sequences from all known variable region classes (Herron, J.N., (1989), supra) and the number of differences in the amino-acids thatmust be retained from the murine were scored. The positions of theseamino acids were obtained from those of the B1312 murine monoclonalantibody, which was chosen to model the humanization of the anti-KC-4antibody.

Based on the these scores, the consensus sequences human frameworksbelonging to groups V_(κ)II and V_(H)III were chosen to receive theanti-KC-4 murine antibody CDRs plus other important amino acids.

Example 15 Identification of Murine-Human anti-KC-4 Antibody Differences

The original murine sequences (anti-KC-4 V_(κ)and V_(H)) were alignedwith their closest human (Human KII or HIII) relatives (see, Example 14above), and the differences in the FR amino acids were noted. In thepresent example, it was intended to be substituted as many amino acidsas possible in going from the murine to the humanized variable consensussequences, leaving the important amino acids intact as described inExamples 14 and 16. The amino acids chosen to be preserved weresubset,of those listed above. Those were selected by analogy to theB1312 sequence. The single exception was the glycine (100) residue ofthe original framework of the variable region of the murine kappa chain,which was retained despite not being encompassed in Table 18 above sinceit was thought that it might contact the variable domain of the heavychain. Such contacts were observed in at least three FAB that lack a glyat this position.

Example 16 Identification of Important Murine anti-KC-4 Antibody AminoAcids

The “important” murine amino acids were chosen for preservation based onthe contacts of a particular amino acid with the CDRs, and with theopposite chains and/or whether their side chains are pointing inwardlyor outwardly. The positions of these “important” amino acids weredetermined based on the examination of the known structures of otherantibodies.

Most of the “important” amino acids were selected on the basis of thestructure of antibody B1312 and according to Tables 2, 3, 4, 5, 6, 8 and9 above.

The final selection of amino acid positions for actual mutation wasattained by comparing the position of all amino acids that arecandidates for mutation with those that are “important” and should bepreserved. Any “important” amino acid position was eliminated from thelist of candidates. Table 19 below shows the amino acids that wereselected for change in the murine sequence to attain the humanizedsequence in the present exemplary analogue.

TABLE 19 Anti-KC-4 Murine Antibody Variable Region Amino Acids Selectedfor Mutation Position KC-4G3 Murine Identity → Consensus Human IdentityLight Chain Variable Region T 14 S T 15 L P 17 D E 18 Q P 45 K Q 74 N K83 L V Heavy Chain Variable Region 13 K Q 19 K R 40 S A 42 E G 44 R G 74A S 61 E Q  82a S N 84 S A 89 M V 110  S T 113  A S

The change N→K at position 74 in the variable light chain knowinglyeliminated an N-linked glycosylation site, which was present in theoriginal murine monolonal antibody.

Example 17 Introduction of Changes in Amino Acid Sequence forHumanization of anti-KC-4 Antibody

The introduction of the changes in the amino acid sequence was conductedas follows. The DNA encoding each humanized variable region wassynthesized in a single polymerase chain reaction (PCR) usingoverlapping oligonucleotides in accordance with the method described byYe et al. (Ye, Q-Z, Johnson, L. L., and Baragi, V., “Gene Synthesis andExpression in E. coli for PUMP, a Human Matrix Metalloproteinase”, BBRC186(1):143-149 (1992)). The sequences of the oligonuclcotides are shownin Table 20 below.

TABLE 20 Primers for Humanization of anti-KC-4 Murine Antibody VariableRegions JA59 CCCGGATCC TTTAAAAGGT GTCCAGTGTG AAGTGCAGAT GGTGGAG TCT G(SEQ. ID No.: 37) J060 GAATTCGGGGC TAGCACTAGA GACAGTGACC AGAGTCCCTTGGCCC CAG (SEQ. ID No.: 38) J061 AGTGCAGATG GTGGAGTCTG GGGGAGGCTTAGTGCAGCCT GGAGGG TCCC TGAGACTCTC CTGTGCAGCC TCTGGATTCG CTTTCAGTAGCTATGCCATG T (SEQ. ID No.: 39) J062 CTTGATAGTA GGCGTAATTA CCACCACTACTAATTTCTGC GACCCA CTCC AGCCCCTTCC CTGGAGCCTG GCGAACCCAA GACATGGCATAGCTACTGAA A (SEQ. ID. No.: 40) J063 TAATTACGCC TACTATCAAG ACACTGTGACGGGCCGATTC ACCATC TCCA GAGACAATTC CAAGAACACC CTGTACCTGC AAATGAACAGTCTGAGGGCT G (SEQ. ID. No.: 41) J064 CCAGAGTCCC TTGGCCCCAG TAAGCAAACCAGGCCGGGAT ACCGTA GTCC TCCCTTGCAC AGTAATACAC GGCCGTGTCC TCAGCCCTCAGACTGTTCAT T (SEQ. ID. No.: 42) J073 GGGAAGCTTG ATATCCACCA TGAAGTTGCCTGTTAGGCTG TTGGTG CTGA TGTTCTGGAT TCCTGC (SEQ. ID. No.: 43) J074AAAGATTCG TCGACTTACG TTTTATTTCC AGCTTGGTCC CCCCTCC GAA CGTGTACGGA ACATGT(SEQ. ID. No.: 44) J075 CTGATGTTCT GGATTCCTGC TTCCAGCAGT GATGTTTTGATGACCC AAAC TCCTCTCTCC CTGCCTGTCA CTCCAGGAGA GCCAGCCTCC ATCTCTTGCA (SEQ.ID. No.: 45) J076 CTGTGGAGAC TGGCCTGGTT TCTGCAGGTA CCATTCTAAA TAGGTGTTTC CATTACTATG TACAATGCTC TGACTAGATC TGCAAGAGAT GGAGGCTGGC (SEQ. ID.No.: 46) J078 CGAACGTGTA CGGAACATGT GAACCTTGAA AGCAGTAATA AATTCC CACATCCTCAGCCT CCACTCTGCT GATCTTGAGT GTGAAATCTG TCCCTGATCC (SEQ. ID. No.:47)

Example 18 Synthesis of Primers for Humanization of anti-KC-4 Antibody

All primers were synthesized on a PCR-Mate EP DNA synthesizer model 391(Applied Biosystems, Foster City, Calif.) using 40 nmole columns, cycle1:63, with Trityl off. None were purified before use. Their sequencesare shown in Table 20 above.

Example 19 Synthesis of anti-KC-4 Humanized Heavy Chains VariableRegions

A mixture of PCR primers was made, where each primer was present at aconcentration of 10 prmole/μd in water.

Four 101 'mer oligonucleotides (JO61, JO62, JO63 and JO64), one 50'mer(JO59) and one 49'mer (JO60), were used for synthesis of the humanizedvariable heavy chain. The oligonucleotides concentration were estimatedusing the formulac=[(A_(2∞)/)30]μg/μl

The PCR amplification conditions were as follows. All reagents as wellas the GeneAmp PCR system 9600 were purchased from Perkin Elmer Cetus.Optimal PCR conditions were determined empirically for each pair ofmutagenic primers. A matrix of conditions varying the concentration ofMgCl₂, mutagenic primer, and template plasmid DNA were set up asfollows. However, the annealing and extension temperatures during PCRmay be varied.

2 μM primer JO59 150 nM each of primers JO61, 62, 63 and 64. 2 μM primerJO60 200 μM each of dGTP, dATP, TTP, and dCTP. 10 mM KCl 20 mM Tris-HClpH 8.8 10 mM (NH₄)₂SO₄ 2 units per 100 μl reaction Vent DNA 0.1% TritonX-100 polymerase (New England Biolabs) 6 mM MgSO₄

Example 20 Hot Start PCR for Humanization of anti-KC-4 Antibody

All the components of the PCR mixture, with the exception of Vent DNApolymerase, were mixed. The mixture was then dispensed in 19 μl aliquotsin 5 PCR tubes. The reason for performing five independent reactions wasto decrease the odds that unwanted mutations be isolated as a result ofnucleotide misincorporation during PCR. The tubes were heated to 95° C.for 5 minutes and then cooled to 72° C. While at that temperature 1 μlof an appropriate Vent DNA polymerase dilution in 1×buffer was added tothe reaction mixture (hot start). The temperature cycling then proceedsas follows.

[(96° C., 6 sec) (55° C., 10 sec) (72° C., 30 sec)] 3 cycles

[(96° C., 5 sec) (60° C., 10 sec) (72° C., 30 sec)] 29 cycles

72° C., 10 min

Example 21 Extra Final Extension for Humanized anti-KC-4 Antibody V_(H)DNA

After cycling, one extra final extension reaction was carried out Extradeoxyribonucleotide triphosphates (to 125 μM) and 1 unit of Vent DNApolymerase were added, and the mixture was heated to 72° C. for 10minutes.

The resulting synthetic DNA fragment was digested with Dral and Nbel andinserted into the same restriction sites an intermediate plasmidconstruct encoding the corresponding murine heavy chain variable regionas described in examples 23 to 25.

Example 22 Synthesis of anti-KC-4 Humanized Light Chain Variable Regions

The light chain variable region (V_(L)) genes were synthesized in asimilar way as described in Examples 22 to 24 above for the heavy chainsignal peptide variable regions. In this case, however, the completesignal peptide and the V_(L) encoding DNA were contained between EroRVand SaII. This DNA was inserted (ligated) into pBluescriptIIKs⁺(Stratagene) as described in examples 23 and 25.

Example 23 Purification of Humanized anti-KC-4 PCR Products

The PCR products were then separated on a 0.8% agarose gel in 1XTAEbuffer and 0.5 μg/ml ethidium bromide. The correct DNA bands werevisualized with UV light (366 nm), excised from the gels and extractedwith the GeneClean kit (Bio 101, La Jolla, Calif.).

Example 24 Ligation of Humanized anti-KC-4 DNA to Plasmids (Reclosure ofPlasmid)

The ligation mixtures consisted of 5 μl extracted DNA, 2 μl 10× ligationbuffer (NEB) 1 μl T4 DNA polymerase (NEB), 12 μl water. The amount ofplasmid DNA may be varied depending of the intensity of the bandextracted from the Gel. Ligations were carried out at room temperaturefor 2 hrs., or alteratively at 14° C. overnight.

Example 25 Transformation and Sequencing of Humanized anti-KC-4 DNA

The reclosed plasmids were then transformed into E. coli utilizing Invalpha F′ competent cells purchased from Invitrogen Corporation, SanDiego Calif. Plasmid DNA was then prepared from a few transformants andsequenced to verify that mutagenesis was successful.

Example 26 Hybrid Plasmid Preparation and Sequencing

Plasmid DNA was then prepared and sequenced to verify that the genesynthesis was successful. The anti-KC-4 humanized DNA sequences for theV_(H) and V_(L) segments are shown in Tables 21 and 22 below.

TABLE 21 Humanized anti-KC-4 Antibody V_(L) DNA sequences anti-KC-4V_(L) FR-HZ ATG AAG TTG CCT GTT AGG CTG TTG GTG CTG ATG TTC TGG ATT CCTGCT TCC AGC AGT GAT GTT TTG ATG ACC CAA ACT CCT CTC TCC CTG CCT GTC ACTCCA GGA GAG CCA GCC TCC ATC TCT TGC AGA TCT AGT CAG AGC ATT GTA CAT AGTAAT GGA AAC ACC TAT TTA GAA TGG TAC CTG CAG AAA CCA GGC CAG TCT CCA CAGCTC CTG ATC TAC AAA GTT TCC ATC CGA TTT TCT GGG GTC CCA GAC AGG TTC AGTGGC AGT GGA TCA GGG ACA GAT TTC ACA CTC AAG ATC AGC AGA GTG GAG GCT GAGGAT GTG GGA ATT TAT TAC TGC TTT CAA GGT TCA CAT GTT CCG TAC ACG TTC GGAGGG GGG ACC AAG CTG GAA ATA AAA C (SEQ. ID. NO.: 48)

TABLE 22 Humanized anti-KC-4 Antibody V_(H) DNA Sequences anti-KC-4V_(H) FR-HZ ATG GAC TTT GGG CTC AGC TTG GTT TTC CTT GTC CTT ATT TTA AAAGGT GTC CAG TGT GAA GTG CAG ATG GTG GAG TCT GGG GGA GGC TTA GTG CAG CCTGGA GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC GCT TTC AGT AGC TATGCC ATG TCT TGG GTT CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTC GCA GAAATT AGT AGT GGT GGT AAT TAC GCC TAC TAT CAA GAC ACT GTG ACG GGC CGA TTCACC ATC TCC AGA GAC AAT TCC AAG AAC ACC CTG TAC CTG CAA ATG AAC AGT CTGAGG GCT GAG GAC ACG GCC GTG TAT TAC TGT GCA AGG GAG GAC TAC GGT ATC CCGGCC TGG TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC TCT AGT (SEQ.ID. No.: 49)

Example 27 Expression of anti-KC-4 Humanized Antibody

Two expression vectors pAG4622 and pAH4604 (Coloma, M. J., et al.(1992), supra) were used that were developed and provided by S. L.Morrison (Dept. of Microbiology and Molecular Genetics, UCLA). Any cDNAencoding a signal peptide and either the variable heavy chain or thevariable light chain can, in principle, be inserted into these vectorsresulting in a construction that encodes an IgG1, K, antibody with humanconstant regions. Synthetic DNA fragments were excised from theirintermediate plasmids (see examples 21 and 22) with either EcoRV and Salto be inserted into pAG4622(light chain vector), or with EcoRV and NbElto be inserted into pAH4640 (heavy chain vector). The restriction andligation reactions necessary to accomplish these operations wereperformed under the conditions stipulated by the enzyme manufacturers(New England Biolabs, Beverly, Mass.). Both the vectors and the insertswere purified from an agarose gel prior to ligation, using the Bio101(La Jolla, Calif.) GeneClean kit (glass beads). The V_(H) and V_(L)regions in the final constructions were sequenced once again to verifythat they were correct. The non-producer mycloma cell line SP2/0-Ag14,ATCC: CRL 1581, (Shulman M., et al. (1978), supra) was transfected withboth plasmid constructions, and antibody producers were isolatedfollowing the recommendations outlined in Coloma et al. (Coloma, M. J.et al. (1992), supra) except that selection was done only for the uptakeof hisD (by adding 5 mM histidinol to the medium and readjusting the pHto 7.4 with NaOH). Usually after ten days, stable transfectant colonieswere established at a frequency of approximately 10⁻³ to 10⁻⁴. Colonieswere then transferred to normal medium (without histidinol). The culturemedia were either Dulbeco's modified Eagle's medium (DME): fetal bovineserum (FBS), 90:10, v/v, or a mixture of DME:RPMI:FBS, 45:45:10, v/v/v.Penicillin and streptomycin were added to prevent bacterial growth.

The supernatants from stable transfectants were assayed for the presenceof the antibodies. This was done by capturing the secreted chimericantibody with a plate-bound goat anti-human-kappa chain antibody anddeveloping with goat anti-human-gamma chain antibody, essentially asdescribed previously (Coloma, M. J., (1992), supra) except that thesecondary antibody was radiolabeled with ¹²⁵I. The supernatants werealso assayed for binding to human milk fat globule (HMFG) as describedpreviously (Ceriani R. L., et al., “Diagnostic Ability of DifferentHuman Milk Fat Globule Antigens in Breast Cancer”, Breast Cancer Res.Treat. 15:161-174 (1990)). HMFG is bound to the microtiter plates asdescribed previously (Ceriani, R. I. (1984), supra). Usually most colonysupernatants were positive by both assays.

Colonies that secrete the highest level of antibody in the supernatants,as determined by these assays, were subcloned and subsequently adaptedto serum-free medium for the purification of antibody. Serum free mediumcontains HL-1 supplement as directed by the manufacturer (Ventrex Labs,Portand, Me.).

Example 28 Half Humanized-Half Chimeric anti-KC-4 Antibody

An anti-KC-4 humanized light chain was paired with an anti-KC-4non-humanized chimeric heavy chain by co-transfection of SP2/0Ag14mycloma cells with hybrid plasmids carrying the respective DNA sequencesand those of a human F_(C). The resulting antibody was named “HuKC4V1”(ATCC No. HB 11454).

In addition, an anti-KC-4 humanized heavy chain was paired with ananti-KC-4 non-humanized chimeric light chain as described in Example 27above. The resulting antibody was named “HuKC4V3” (ATCC No. HB 11456).

Example 29 Fully Humanized anti-KC-4 Antibody

An anti-KC-4 fully humanized antibody was prepared by pairing fullyhumanized anti-KC-4 light and heavy chains by co-transfection asdescribed in Example 27 above. The fully humanized versions is named“HuKC4V2” (ATCC No. HB 11455).

Example 30 Determination of Affinity Constants for Fully Humanizedanti-KC-4 Antibody

The secreted fully humanized antibody (HuKC4V2) was purified fromculture supernatants using a Sepharose 4B -protein A column (Bio-Rad,Richmond, Calif.) as described by Ey et al. (Ey, P. L, et al. (1978),supra). Microtiter plates (Dynatech, Chantilly, Va.) were prepared asdescribed by Ceriani et al. (Ceriani R. L., et al. (1992), supra) usingsuccessive layers of methylated BSA, glutaraldehyde,anti-β-galactosidase and the bacterial fusion protein 11-2 (a hybrid ofβ-galactosidase and human mammary mucin). Each well contained 388 mg ofthe 11-2 fusion protein. To each well were added 25 μl ¹²⁵I-KC-4 in RIAbuffer (10% bovine calf serum, 0.3% triton X-100, 0.05% sodium azide pH7.4, in phosphate buffer saline) and compete with 25 μl of eitherunlabeled murine or chimeric antibody in RIA buffer at the finalconcentrations of 130 pM, 850 pM, 1.3 nM, 4 nM, and 13 nM). Iodinationswere performed with ¹²⁵I (17 Ci/mg, Nordion International). 50 μganti-KC-4 monoclonal antibody (Coulter, Hialeah, Fla.) were labeled at aspecific activity of 9.56 mCi/mg using the chloramine T method asdescribed previously by Ceriani et al. (Ceriani R. L., et al., (198),supra).

The antibody-antigen affinity constants were determined by taking thereciprocal of the concentration of competing unlabeled monoclonalantibody that produced 50% binding as described by Sheldon et al.(Sheldon K., et al. (1987), supra).The protocol used to determineaffinity constants was as described above except that in each case, anunlabeled antibody competed for binding to the antigen against the sameradiolabeled antibody. The fully humanized antibody was shown to competeas well as anti-KC-4 murine antibody against radiolabeled anti-KC-4murine antibody for binding to the KC-4G3 antigen.

Polyacrylamide gel electrophoresis was performed to insure that theantibody chains migrated as expected. The affinity binding constants ofthe murine, chimeric, half humanized and humanized antibodies weredetermined in independent competition assays. The binding affinities ofthe murine and anti-KC-4 and HuKC4V2 antibodies for the KC-4G3 antigenwere determined to be similar.

Example 31 Histochemical Specificity of Fully Humanized Antibody

Immunohistochemical staining using the immunoperoxidase technique ofconsecutive human breast carcinoma tissue sections was used as a test toverify that the analogue antibodies retain the affinity for the KC-4G3carcinoma antigen of the murine antibody. Breast carcinoma tissuesections were stained with the supernatant of the KC-4 murine and fullyhumanized transfected cells using the Vectastain ABC method (VectorLabs, Burlingame, Calif.). Both antibodies showed strong stainingpatterns.

The following Table 23 shows the results of the immunoperoxidasestaining of five human breast carcinomas with either the standardanti-KC-4G3 murine or the fully humanized antibodies. Both stained thesame tissues at a comparative level.

TABLE 23 Immunoperoxidase Staining of Human Breast Carcinoma TissueSections with Murine and Fully Humanized anti-KC-4 Antibodies BreastTumor Murine Antibody Fully Humanized Antibody 1 ++ ++ 2 +++ +++ 3 − − 4++ ++ 5 +++ +++

Example 32 Binding to HMFG of Half Humanized and Fully Humanizedanti-KC-4 Antibodies

Tissue culture supernatants from transfections of all three anti-KC-4variants of the humanized antibody were shown to bind the human milk fatglobule (HMFG) as determined by radio-immunodetections.

Example 33 Half Humanized and Fully Humanized anti-KC-4 Antibodies Bindto Goat anti-Human κ or γ Antibodies

Tissue culture supernatants from transfections of all three variants ofthe anti-KC-4 humanized antibody were shown to bind in sandwichradioimmunodetections to both goat anti-human kappa chain antibody boundto microtiterplate wells (750 ng/well), and to radio-iodinated¹²⁵I-labeled goat anti-human gamma chain antibodies.

The results of these sandwich assays demonstrate that both chains of thehumanized antibodies indeed possess human kappa and gamma constantregions.

Example 34 Deduced Amino Acid Sequence of Humanized anti-KC-4 VariableLight and Heavy Chains

The amino acid sequences of the light and heavy chains of the analoguehumanized antibody are shown in Tables 24 and 25 below. The actual aminoacid sequences may be varied either to increase affinity for the antigenor to decrease immunogenicity in humans. Numerous variants of thissequence may be engineered in accordance with the invention.

TABLE 24 Humanized anti-KC-4 Antibody V_(L) Analogue Sequence anti-KC-4V_(L) FR-HZ MKLPVRLLVL MFWIPASSSD VLMTQTPLSL PVTPGEPASI SCRSSQSIVHSNGNTYLEWY LQKPGQSPQL LIYKVSIRFS GVPDRFSGSG SGTDFTLKIS RVEAEDVGIYYCFQGSHVPY TFGGGTKLEI K (Seq. ID No: 50)

TABLE 25 Humanized anti-KC-4 Antibody V_(H) Analogue sequence anti-KC-4V_(H) FR-HZ MDFGLSLVFL VLILKGVQCE VQMVESGGGL VQPGGSLRLS CAASGFAFSSYAMSWVRQAP GKGLEWVAEI SSGGNYAYYQ DTVTGRFTIS RDNSKNTLYL QMNSLRAEDTAVYYCAREDY GIPAWFAYWG QGTLVTVSS (Seq. ID No: 51)

Example 35 Hybridoma Cell Deposits

The following cell lines were deposited as present examples of the bestmode of the invention. The hybridoma cell line expressing the anti-KC4murine-human chimeric antibody was deposited with the ATCC on Nov. 13,1992 under the Budapest Treaty, and has been assigned Accession No. HB11201 (Chimeric anti-KC-4 1E8). The hybridoma cell lines expressing theanti-KC-4 fully humanized antibody (huKC4V2), and the half humanizedanti-KC-4 antibodies (huKC4V1 and huKC4V3) were deposited with the ATCCon Sep. 23, 1993 and have been assigned Accession Nos. HB 11455(Humanized HuKC-4 V2), HB 11454 (Half Humanized HuKC4V1), and HB 11456(Half Humanized HuKC4V3).

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

1. A modified chimeric humanized anti-KC-4 antibody which selectivelybinds to the human KC-4 antigen, comprising (1) the humanized variableregions of the light and heavy chains of an anti-KC-4 murine antibodyand (2) light and heavy chain constant regions of a human antibody,wherein the amino acid sequence of the variable regions comprises SEQ IDNOS: 50 and SEQ ID NO:
 51. 2. The antibody of claim 1, wherein 12 aminoacids in the light chain framework regions (FRs) are substituted and 7amino acids in the heavy chain FRs are substituted.
 3. The antibody ofclaim 2, wherein said amino acids in said framework regions are aminoacids present in equivalent positions in antibodies of species otherthan murine.
 4. An antibody according to claim 1, further comprising atleast one glycosyl residue attached thereto.
 5. A composition of matter,comprising the antibody of claim 1 and a carrier.
 6. A cancer diagnostickit, comprising the composition of claim 5, wherein said carrier is apharmaceutically acceptable carrier for in vivo administration, andwherein said antibody is in radiolabeled form.
 7. A cancer therapy kit,comprising the composition of claim 4 in a pharmaceutically-acceptableform, wherein said antibody is in radiolabeled form.
 8. An in vitrocancer diagnostic kit, comprising the antibody of claim 1; and a solidsupport.
 9. The in vitro kit of claim 8, further comprising one or moreheterologous immunoglobulins that selectively bind to the constantregion of the anti-KC-4 antibody.
 10. The in vitro kit of claim 9,further comprising a label selected from the group consisting of aradioisotope, an enzyme, a phosphorescent molecule, and a fluorescentmolecule, wherein said label is conjugated to said antibody of saidimmunoglobulin.
 11. A hybridoma cell expressing the antibody of claim 1.12. The hybridoma cell of claim 11, having the ATCC Accession No. HB11455 (HuKC4V2).
 13. A composition, comprising the hybridoma cell ofclaim 11, and a diluent or a carrier.
 14. A method of determining thepresence of cancer cells in a tissue or a blood sample thereof,comprising contacting a tissue or a blood sample with the anti-KC-4antibody of claim 1 or an antigen binding fragment thereof, anddetecting the presence of an immune complex formed between saidanti-KC-4 antibody and the KC-4 antigen of said tissue or said bloodsample thereof.
 15. A method according to claim 14, wherein the tissueis excised from a subject prior to being contacted with the anti-KC-4antibody, and wherein said contacting and detecting steps are carriedout in vitro.
 16. A method according to claim 15, further comprisingcomparing results obtained by analyzing said formed complexes with astandard cut-off value determined by comparing results obtained fornormal and cancer patients' samples wherein a result above a cut-offvalue is indicative of cancer.
 17. A method of imaging a cancer,comprising administering a pharmaceutically-acceptable compositioncomprising an effective imaging amount of the anti-KC-4 antibody ofclaim 1 or an antigen binding fragment thereof, in radiolabeled form, toa subject suspected of having a primary or metastasized cancer; anddetecting any binding of the labeled antibody to cancer cells in thesubject by external imaging.
 18. A method of treating cancer in asubject, comprising administering to a subject in need of treatment aneffective therapeutic amount of the antibody of claim 1, wherein theantibody is conjugated to a therapeutic agent.
 19. A method of purgingcancer cells from a biological sample, comprising contacting abiological sample obtained from a subject suspected of having cancerwith the antibody of claim 1 and allowing said antibody to bind the KC-4antigen associated with cancer cells in the sample to form an immunecomplex between said antibody and the antigen of said biological sample;and removing said immune complex from the biological sample.
 20. Amethod according to claim 14, wherein said tissue sample is prepared forimmunohistochemical analysis.
 21. A method of purging cancer cells froma biological sample and replenishing the purged sample to a subject,comprising the method of claim 19, wherein said method further comprisesreintroducing the purged biological sample to the subject.